Compositions and methods for treating cancer

ABSTRACT

Use of a CXCR4 antagonistic peptide and an immune-check point regulator in the treatment of cancer is provided. Accordingly there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof; and a therapeutically effective amount of a PD1 antagonist, a PDL-1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a TIM-3 antagonist, a KIR antagonist, an IDO antagonist, an OX40 agonist, a CD137 agonist, a CD27 agonist, a CD40 agonist, a GITR agonist, a CD28 agonist or an ICOS agonist, thereby treating the cancer in the subject. Also provided are pharmaceutical compositions and articles of manufacture.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/571,505 filed on Nov. 3, 2017, which is a National Phase of PCTPatent Application No. PCT/IL2016/050764 having International FilingDate of Jul. 14, 2016, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application Nos. 62/291,039 filed onFeb. 4, 2016, 62/259,182 filed on Nov. 24, 2015, 62/291,006 filed onFeb. 4, 2016 and 62/193,201 filed on Jul. 16, 2015. The contents of theabove applications are all incorporated by reference as if fully setforth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 82481SequenceListing.txt, created on Apr. 21,2020, comprising 39,896 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference. The sequencelisting submitted herewith is identical to the sequence listing formingpart of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof treating cancer and, more particularly, but not exclusively, to theuse of a CXCR4 antagonistic peptide and an immune-check point regulatorin the treatment of cancer.

Cancer is the second leading cause of death in the U.S.A. The estimatesfor 2014 are that approximately 585,000 people will die of cancer and1.6 million new cases will be diagnosed (American Cancer Society, CancerFacts & FIGS. 2014).

For early stage cancers, surgical removal is a very effective treatment.

However, for more advanced cases and non-solid hematologicalmalignancies, standard, non-specific cancer treatments such aschemotherapy and radiotherapy are typically used. These treatmentsaffect many healthy cells and result in elevated toxicity and effectivein only a minor percentage of treated individuals. Moreover, evenindividuals that initially respond to therapy are at risk for relapses,and often develop resistance.

Significant progress in understanding the underlying principles of tumorbiology as well as the basic mechanisms of the immune response to cancerhave led to the development of new immunotherapies aimed at employingthe adaptive immune system to eradicate cancer with enhanced efficacyand reduced toxicity. Until recently, cancer immunotherapy had focusedon approaches that enhance anti-tumor immune responses byadoptive-transfer of activated effector cells, immunization againstrelevant antigens, or providing non-specific immune-stimulatory agentssuch as cytokines. In the past decade, however, intensive efforts todevelop specific immune checkpoint pathway inhibitors and co-stimulatorypathway activators have begun to provide new immunotherapeutics fortreating cancer. Thus, for example, ipilimumab (YERVOY®), an antibodythat binds to and inhibits the immune regulatory protein CTLA-4 andpembrolizumab (KEYTRUDA®), an antibody that binds to and inhibits theimmune regulatory protein PD1, have been approved by the United StatesFood and Drug Administration for the treatment of melanoma. Otheranti-PD-1 antibodies (such as Nivolumab) have also shown efficacy inother solid tumors such as non-small-cell lung cancer, and renal-cellcancer [Topalian et al. N Engl J Med. (2012) 366(26):2443-54].

4F-benzoyl-TN14003 (also known as BKT140, hereinafter BL-8040), is a14-residue bio stable synthetic peptide developed as a specific CXCR4antagonist. It has been shown that BL-8040 binds the CXCR4 receptor withhigh affinity and long receptor occupancy. Studies in mice demonstratedthat a single BL-8040 injection mobilized long term repopulating stemcells sufficient for transplantation. [Abraham M et al., Stem Cells(2007); 25:2158-66] Results from a study in multiple myeloma patientsshowed that combined treatment of BL-8040 and G-CSF enabled thecollection of high number of CD34+hematopoietic stem/progenitor cells(HSPC) in a single aphaeresis procedure [Peled A et al. Clin Cancer Res;(2013) 20(2); 469-79].

In addition, BL-8040 was found to be toxic against several tumors suchas myeloid leukemia, hematopoietic tumors and non-small cell lung cancer(International Patent Application No. IL2014/050939 and InternationalPatent Application Publication Nos. WO2013/160895 and WO2008/075370.

Additional Background Art Includes:

International Patent Application Publication No. WO2014/155376;

International Patent Application Publication No. WO2012/095849;

International Patent Application Publication No. WO2002/20561;

International Patent Application Publication No. WO2004/020462;

International Patent Application Publication No. WO2008/075369;

International Patent Application Publication No. WO2008/075371;

International Patent Application Publication No. WO2010/146578;

International Patent Application Publication No. WO2010/146584;

International Patent Application Publication No. WO2003/072599;

International Patent Application Publication No. WO2015/019284;

U.S. Patent Application Publication No. 2012/0082687; and

Chen et al. HEPATOLOGY (2015) 61: 1591-1602.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aPD1 antagonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aPD-L1 antagonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCTLA-4 antagonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aLAG-3 antagonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aTIM-3 antagonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aKIR antagonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aIDO antagonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aOX40 agonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCD137 agonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCD27 agonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCD40 agonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aGITR agonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCD28 agonist, thereby treating the cancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aICOS agonist, thereby treating the cancer in the subject.

According to some embodiments of the invention, the (a) is effectedprior to (b).

According to some embodiments of the invention, the (a) is effectedfollowing (b).

According to some embodiments of the invention, the (a) is effectedconcomitantly with (b).

According to some embodiments of the invention, (a) is effected multipletimes.

According to some embodiments of the invention, (b) is effected multipletimes.

According to some embodiments of the invention, the (a) and the (b) areeffected sequentially.

According to some embodiments of the invention, the (a) is effected at adose of 0.5-1 mg/kg.

According to some embodiments of the invention, the (a) is administeredsubcutaneously.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a PD1 antagonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a PD-L1 antagonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof; and a CTLA-4 antagonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a LAG-3 antagonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a TIM-3 antagonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a KIR antagonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a IDO antagonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a OX40 agonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD137 agonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD27 agonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD40 agonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a GITR agonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD28 agonist.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for use intreating cancer, comprising a packaging material packaging a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a ICOS agonist.

According to some embodiments of the invention, the peptide and theantagonist are in separate formulations.

According to some embodiments of the invention, the peptide and theantagonist are in a co-formulation.

According to some embodiments of the invention, the peptide and theagonist are in separate formulations.

According to some embodiments of the invention, the peptide and theagonist are in a co-formulation.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a PD1 antagonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a PD-L1 antagonist; anda pharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof; and a CTLA-4 antagonist;and a pharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a LAG-3 antagonist; anda pharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a TIM-3 antagonist; anda pharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a KIR antagonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a IDO antagonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a OX40 agonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a CD137 agonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a CD27 agonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a CD40 agonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a GITR agonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a CD28 agonist; and apharmaceutically acceptable carrier or diluent.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as activeingredients a peptide having an amino acid sequence as set forth in SEQID NO: 1 or an analog or derivative thereof and a ICOS agonist; and apharmaceutically acceptable carrier or diluent.

According to some embodiments of the invention, the antagonist is anantibody.

According to some embodiments, the method of treating cancer furthercomprises administering a vaccine and optionally wherein the vaccine isan HPV vaccine.

According to some embodiments of the invention, the antagonist is asmall molecule.

According to some embodiments of the invention, the antagonist is apeptide.

According to some embodiments of the invention, the agonist is anantibody.

According to some embodiments of the invention, the agonist is a smallmolecule.

According to some embodiments of the invention, the agonist is apeptide.

According to some embodiments of the invention, the analog or derivativehas an amino acid sequence as set forth in formula (I) or a saltthereof:

(I) 1  2  3  4   5  6  7  8 9 10 11 12  13 14 A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁

wherein:

A₁ is an arginine, lysine, ornithine, citrulline, alanine or glutamicacid residue or a N-α-substituted derivative of these amino acids, or A₁is absent;

A₂ represents an arginine or glutamic acid residue if A₁ is present, orA₂ represents an arginine or glutamic acid residue or a N-α-substitutedderivative of these amino acids if A₁ is absent;

A₃ represents an aromatic amino acid residue;

A₄, A₅ and A₉ each independently represents an arginine, lysine,ornithine, citrulline, alanine or glutamic acid residue;

A₆ represents a proline, glycine, ornithine, lysine, alanine,citrulline, arginine or glutamic acid residue;

A₇ represents a proline, glycine, ornithine, lysine, alanine, citrullineor arginine residue;

A₈ represents a tyrosine, phenylalanine, alanine, naphthylalanine,citrulline or glutamic acid residue;

A₁₀ represents a citrulline, glutamic acid, arginine or lysine residue;

A₁₁ represents an arginine, glutamic acid, lysine or citrulline residuewherein the C-terminal carboxyl may be derivatized;

and the cysteine residue of the 4-position or the 13-position can form adisulfide bond, and the amino acids can be of either L or D form.

According to some embodiments of the invention, the peptide is selectedfrom the group consisting of SEQ ID NOs: 1-72.

According to some embodiments of the invention, the peptide is as setforth in SEQ ID NO: 1.

According to some embodiments of the invention, the cancer is a solidtumor cancer.

According to some embodiments of the invention, the solid tumor isselected from the group consisting of lung cancer, glioma, colon cancer,ovarian cancer, renal cancer, melanoma cancer, hepatocellular cancer,gastric or stomach cancer, glioblastoma, cervical cancer, bladdercancer, breast cancer, colorectal cancer, prostate cancer, thyroidcancer, head and neck and pancreatic cancer.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof treating cancer and, more particularly, but not exclusively, to theuse of a CXCR4 antagonistic peptide and an immune-check point regulatorin the treatment of cancer.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Treatment of most types of cancer involves cytotoxic treatments such aschemotherapy and radiotherapy that may at least in part affect manyhealthy cells and thus result in elevated toxicity. In addition, thesetreatments are effective in only a small percentage of cancer affectedpatients. Immunotherapy strategies for cancer therapy, aiming atharnessing the immune system to fight cancer, include cytokines,monoclonal antibodies against tumor cells or immune regulatorymolecules, cancer vaccines as well as cell-based therapies such asadoptive transfer of ex-vivo activated T cells and natural killer (NK)cells.

4F-benzoyl-TN14003 (SEQ ID NO: 1, also known as BKT140, hereinafterBL-8040) is a CXCR4 peptide antagonist. It has been shown that BL-8040induces mobilization of CD34+hematopoietic stem/progenitor cells (HSPC)that can be further used for transplantation. In addition, BL-8040 wasfound to be toxic against several tumors such as myeloid leukemia,hematopoietic tumors and non-small cell lung cancer.

While reducing the present invention to practice, the present inventorshave found that in-vivo administration of BL-8040 induces rapidmobilization of a variety of immune cells including immaturestem/progenitor cells as well as fully differentiated T cells and NKcells. The present findings therefore can be harnessed to the use ofBL-8040 to induce the mobilization and dissemination of ImDCs and Teffector and memory cells into tumor sites and thus can augment theanti-tumor effect of immunotherapeutics.

Consequently, the present teachings and the protocols presented inExample 1, suggest the use of a peptide having an amino acid sequence asset forth in SEQ ID NO: 1 or an analog or derivative thereof incombination with several combinations of immune-check point regulatorsfor the treatment of cancer.

The terms “treating” or “treatment” refers to inhibiting, preventing orarresting the development of a pathology (e.g. cancer) and/or causingthe reduction, remission, or regression of a pathology. Those of skillin the art will understand that various methodologies and assays can beused to assess the development of a pathology, and similarly, variousmethodologies and assays may be used to assess the reduction, remissionor regression of a pathology.

As used herein the phrase “subject in need thereof” refers to amammalian male or female subject (e.g., human being) who is diagnosedwith cancer. In a specific embodiment, this term encompasses individualswho are at risk to develop cancer. Veterinary uses are alsocontemplated. The subject may be of any gender or at any age includingneonatal, infant, juvenile, adolescent, adult and elderly adult.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth.

Cancers which can be treated by the method of this aspect of someembodiments of the invention can be any solid or non-solid cancer and/orcancer metastasis.

According to a specific embodiment, the cancer is a solid tumor.According another specific embodiment, the cancer is a non-solid tumor.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), melanoma cancer, cancer of theperitoneum, hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; acute myeloblastic leukemia; Multiple Myeloma; andpost-transplant lymphoproliferative disorder (PTLD), as well as abnormalvascular proliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. Preferably, thecancer is selected from the group consisting of breast cancer,colorectal cancer, rectal cancer, non-small cell lung cancer,non-Hodgkins lymphoma (NHL), acute lymphoblastic leukemia (ALL); chronicmyeloblastic leukemia (CML); acute myeloblastic leukemia (AML); renalcell cancer, prostate cancer, liver cancer, pancreatic cancer,soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head andneck cancer, melanoma, ovarian cancer, mesothelioma, and multiplemyeloma. The cancerous conditions amenable for treatment of theinvention include metastatic cancers.

According to specific embodiments the cancer is selected from the groupconsisting of lung cancer, glioma, colon cancer, ovarian cancer, renalcancer, melanoma cancer, hepatocellular cancer, gastric or stomachcancer, glioblastoma, cervical cancer, bladder cancer, breast cancer,colorectal cancer, prostate cancer, thyroid cancer, head and neck andpancreatic cancer.

According to specific embodiments, the cancer is selected from the groupconsisting of lung cancer, glioma, colon cancer and pancreatic cancer.

According to other specific embodiments, the cancer is selected from thegroup consisting of multiple myeloma and leukemia.

As used herein, the term “peptide” encompasses native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), as well as peptoids and semipeptoids which are peptideanalogs, which may have, for example, modifications rendering thepeptides more stable while in a body or more capable of penetrating intocells.

According to a specific embodiment, the peptide is no more than 100amino acids in length. According to a specific embodiment, the peptideis 5-100 amino acids in length. According to a specific embodiment, thepeptide is 5-50 amino acids in length. According to a specificembodiment, the peptide is 5-20 amino acids in length. According to aspecific embodiment, the peptide is 5-15 amino acids in length.According to a specific embodiment, the peptide is 10-20 amino acids inlength. According to a specific embodiment, the peptide is 10-15 aminoacids in length.

As used herein the term “peptide having an amino acid sequence as setforth in SEQ ID NO: 1 or an analog or derivative thereof” refers to4F-benzoyl-TN14003 (SEQ ID NO: 1, also known as BKT140, hereinafterBL-8040) peptide and functional analogs or derivatives thereof. Thepeptides of the present invention are structurally and functionallyrelated to the peptides disclosed in patent applications WO2002/020561and WO2004/020462, also known as “T-140 analogs”, as detailedhereinbelow. The peptide of the present invention is aCXCR4-antagnoistic peptide i.e. it reduces CXCR-4 activation by at least10% as compared to same in the absence of the peptide antagonist.According to a specific embodiment the peptide antagonist is acompetitive inhibitor. According to a specific embodiment the peptideantagonist is a non-competitive inhibitor.

According to specific embodiments, a functional CXCR4 antagonisticpeptide, as used herein, is capable of inducing mobilization anddissemination of ImDCs, NK cells, B cells, monocytes/macrophages and Teffector and memory cells into a tumor of a subject upon administration.

According to other specific embodiments, a functional CXCR4 antagonisticpeptide, as used herein, is capable of enhancing an immune-response to atumor.

In various particular embodiments, the peptide analog or derivative hasan amino acid sequence as set forth in the following formula (I) or asalt thereof:

(I) 1  2  3  4   5  6  7  8 9 10 11 12  13 14 A₁-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-A₈-A₉-A₁₀-Cys-A₁₁

wherein:

A₁ is an arginine, lysine, ornithine, citrulline, alanine or glutamicacid residue or a N-α-substituted derivative of these amino acids, or A₁is absent;

A₂ represents an arginine or glutamic acid residue if A₁ is present, orA2 represents an arginine or glutamic acid residue or a N-α-substitutedderivative of these amino acids if A₁ is absent;

A₃ represents an aromatic amino acid residue;

A₄, A₅ and A₉ each independently represents an arginine, lysine,ornithine, citrulline, alanine or glutamic acid residue;

A₆ represents a proline, glycine, ornithine, lysine, alanine,citrulline, arginine or glutamic acid residue;

A₇ represents a proline, glycine, ornithine, lysine, alanine, citrullineor arginine residue;

A₈ represents a tyrosine, phenylalanine, alanine, naphthylalanine,citrulline or glutamic acid residue;

A₁₀ represents a citrulline, glutamic acid, arginine or lysine residue;

A₁₁ represents an arginine, glutamic acid, lysine or citrulline residuewherein the C-terminal carboxyl may be derivatized;

and the cysteine residue of the 4-position or the 13-position can form adisulfide bond, and the amino acids can be of either L or D form.

Exemplary peptides according to formula (I) are peptides having an aminoacid sequence as set forth in any one of SEQ ID NOs: 1-72, as presentedin Table 1 hereinbelow.

TABLE 1 T-140 and currently preferred T-140 analogs SEQ ID Analog NO:Amino acid sequence 4F-benzoyl-  14F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-TN14003 NH₂ AcTC14003  2Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH AcTC14005 3 Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OHAcTC14011  4Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH AcTC14013 5 Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OHAcTC14015  6Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH AcTC14017  7 Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OHAcTC14019  8Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-OH AcTC14021 9 Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OHAcTC14012 10Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTC14014 11Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH₂AcTC14016 12Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTC14018 13Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTC14020 14Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-NH₂AcTC14022 15Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH₂ TE1400116 H-DGlu-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14002 17 H-Arg-Glu-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14003 18 H-Arg-Arg-Nal-Cys-Tyr-Glu-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14004 19 H-Arg-Arg-Nal-Cys-Tyr-Arg-Glu-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14005 20 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-OHTE14006 21 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Glu-Cit-Cys-Arg-OHTE14007 22 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Glu-OHTE14011 23H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TE1401224 H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14013 25H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TE1401426 H-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14015 27H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH₂ TE1401628 H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH₂AcTE14014 29Ac-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTE14015 30Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH₂AcTE14016 31Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH₂ TF1: 32Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTE14011 TF2: guanyl- 33guanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 TF3: TMguanyl- 34TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 TF4: TMguanyl- 35TMguanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 (2-14) TF5: 4F- 364F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-benzoyl- NH₂ TE14011 TF6: 2F- 372F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-benzoyl- NH₂ TE14011 TF7: APA- 38APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 (2-14) TF8: desamino- 39desamino-R-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂R-TE14011 (2- 14) TF9: guanyl- 40Guanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 (2-14) TF10: succinyl- 41succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 (2-14) TF11: glutaryl- 42glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TE14011 (2-14) TF12: 43deaminoTMG-APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-deaminoTMG- NH₂ APA-TE14011 (2-14) TF15: H-Arg- 44R-CH2-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH2 CH2NH-RTE14011 (2- 14) TF17: TE14011 45H-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH2 (2-14) TF18:46 TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂TMguanyl- TC14012 TF19: ACA- 47ACA-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TC14012TF20: ACA- 48ACA-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH T140TZ14011 49H-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ AcTZ1401150 Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTN14003 51Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂AcTN14005 52Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂4F-benzoyl- 534F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-TN14011-Me NHMe 4F-benzoyl- 544F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-TN14011-Et NHEt 4F-benzoyl- 554F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-TN14011-iPr NHiPr 4F-benzoyl- 564F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-TN14011- tyramine tyramine TA14001 57H-Ala-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14005 58H-Arg-Arg-Nal-Cys-Tyr-Ala-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14006 59H-Arg-Arg-Nal-Cys-Tyr-Arg-Ala-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14007 60H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DAla-Pro-Tyr-Arg-Cit-Cys-Arg-OH TA14008 61H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Ala-Tyr-Arg-Cit-Cys-Arg-OH TA14009 62H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Ala-Arg-Cit-Cys-Arg-OH TA14010 63H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Ala-Cit-Cys-Arg-OH TC14001 64H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC14003 65H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TN14003 66H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ TC1400467 H-Arg-Arg-Nal-Cys-Tyr-Arg-Cit-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1401268 H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂ T-14069 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1401170 H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1400571 H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH TC1401872 H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH₂

According to a specific embodiment, in each one of SEQ ID NOs: 1-72, twocysteine residues are coupled in a disulfide bond.

In another embodiment, the analog or derivative has an amino acidsequence as set forth in SEQ ID NO: 65(H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;TC14003).

In another embodiment, the peptide used in the compositions and methodsof the invention consists essentially of an amino acid sequence as setforth in SEQ ID NO: 1. In another embodiment, the peptide used in thecompositions and methods of the invention comprises an amino acidsequence as set forth in SEQ ID NO: 1. In another embodiment, thepeptide is at least 60%, at least 70% or at least 80% homologous to SEQID NO: 1. In another embodiment, the peptide is at least 90% homologousto SEQ ID NO: 1. In another embodiment, the peptide is at least about95% homologous to SEQ ID NO: 1. Each possibility represents a separateembodiment of the present invention.

In various other embodiments, the peptide is selected from SEQ ID NOs:1-72, wherein each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the peptide has an amino acid sequence as setforth in any one of SEQ ID NOs: 1-4, 10, 46, 47, 51-56, 65, 66, 68, 70and 71. In another embodiment, the peptide has an amino acid sequence asset forth in any one of SEQ ID NOs: 4, 10, 46, 47, 68 and 70. In anotherembodiment, the peptide has an amino acid sequence as set forth in anyone of SEQ ID NOs: 1, 2, 51, 65 and 66. In another embodiment, thepeptide has an amino acid sequence as set forth in any one of SEQ IDNOs: 53-56.

In an embodiment, the peptide has an amino acid sequence as set forth inSEQ ID NO: 1. According to a specific embodiment, the peptide is as setforth in SEQ ID NO: 1. In another embodiment, the peptide has an aminoacid sequence as set forth in SEQ ID NO: 2. In another embodiment, thepeptide has an amino acid sequence as set forth in SEQ ID NO: 51. Inanother embodiment, the peptide has an amino acid sequence as set forthin SEQ ID NO: 66.

According to a specific embodiment the peptide is as set forth in SEQ IDNO: 1 and any embodiment described herein should be read as ifspecifically reading over this peptide.

The peptides of some embodiments of the invention may be synthesized byany techniques that are known to those skilled in the art of peptidesynthesis. For solid phase peptide synthesis, a summary of the manytechniques may be found in J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J.Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, AcademicPress (New York), 1973. For classical solution synthesis see G. Schroderand K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.

In general, these methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then either be attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the final peptidecompound. By simple modification of this general procedure, it ispossible to add more than one amino acid at a time to a growing chain,for example, by coupling (under conditions which do not racemize chiralcenters) a protected tripeptide with a properly protected dipeptide toform, after deprotection, a pentapeptide and so forth. Furtherdescription of peptide synthesis is disclosed in U.S. Patent No.6,472,505.

Large scale peptide synthesis is described by Andersson Biopolymers2000;55(3):227-50.

According to specific embodiments, the CXCR4 antagonistic peptide isadministered to the subject in combination with one or more white bloodcell mobilizing agents. For example, the peptide may be administered insequential or concomitant combination with one or more other growthfactors or cytokines that affect mobilization such as, but not limitedto, G-CSF, GM-CSF and SCF.

As used herein the term “immune-check point regulator” refers to amolecule that modulates the activity of one or more immune-check pointproteins in an agonistic or antagonistic manner resulting in recruitmentof an immune cell to elicit an immune activity against a cancer cell.

According to specific embodiments, the immune-check point regulatormodulates the activity of a specific immune-check point protein with nocross reactivity with other immune-check point proteins.

According to other specific embodiments, the immune-check pointregulator modulates the activity of at least 2, at least 3, at least 4immune-check point proteins.

According to specific embodiments the immune-check point regulator bindsdirectly the immune-check point protein.

According to other specific embodiments, the immune-check pointregulator indirectly binds the immune-check point protein through anintermediary molecule.

As used herein the term “activation” refers to the process ofstimulating an immune cell (e.g. T cell, NK cell, B cell) that resultsin cellular proliferation, maturation, cytokine production and/orinduction of regulatory or effector functions.

As used herein the term “immune-check point protein” refers to anantigen independent protein that modulates an immune cell response (i.e.activation or function). Immune-check point proteins can be eitherco-stimulatory proteins [i.e. positively regulating an immune cellactivation or function by transmitting a co-stimulatory secondary signalresulting in activation of an immune cell] or inhibitory proteins (i.e.negatively regulating an immune cell activation or function bytransmitting an inhibitory signal resulting in suppressing activity ofan immune cell).

According to specific embodiments, the immune-check point proteinregulates activation or function of a T cell. Numerous checkpointproteins are known in the art and include, but not limited to, PD1,PDL-1, CTLA-4, CD80, LAG-3, TIM-3, KIR, IDO, OX40, OX4OL, CD137 (4-1BB),4-1BBL, CD27, CD70, CD40, CD4OL, GITR, CD28, CD86, and ICOS (CD278),ICOSL.

Methods of determining signaling of a stimulatory or inhibitory signalare well known in the art and include, but are not limited to, bindingassay using e.g. BiaCore, HPLC or flow cytometry, enzymatic activityassays such as kinase activity assays, and expression of moleculesinvolved in the signaling cascade using e.g. PCR, Western blot,immunoprecipitation and immunohistochemistry. Additionally oralternatively, determining transmission of a signal (co-stimulatory orinhibitory) can be effected by evaluating immune cell activation orfunction. Methods of evaluating immune cell activation or function arewell known in the art and include, but are not limited to, proliferationassays such as BRDU and thymidine incorporation, cytotoxicity assayssuch as chromium release, cytokine secretion assays such asintracellular cytokine staining ELISPOT and ELISA, expression ofactivation markers such as CD25, CD69 and CD69 using flow cytometry.

According to specific embodiments, determining the signaling activity iseffected in-vitro or ex-vivo e.g. in a mixed lymphocyte reaction (MLR).

For the same culture conditions the signaling activity or the immunecell activation or function are generally expressed in comparison to thesignaling, activation or function in a cell of the same species but notcontacted with the immune-check point regulator or contacted with avehicle control, also referred to as control.

Depending on the immune-check point protein (i.e. co-stimulatory orinhibitory) the immune-check point regulator can be an agonist orantagonist.

According to specific embodiment the immune-check point regulator is anantagonist.

As used herein the term “antagonist” refers to a molecule that preventsand/or inhibits the biological function and/or expression of animmune-check point protein.

According to specific embodiments, the antagonist prevents and/orinhibits the suppressive effect of an immune-check point protein on animmune cell (e.g. T cells).

According to specific embodiments, the antagonist prevents and/orinhibits signaling to an immune cell (e.g. T cell) by an immune-checkpoint protein.

The molecule may be a reversible or an irreversible antagonist.

According to specific embodiments, the antagonist completely preventsthe biological function (e.g. signal transduction) of the immune-checkpoint protein.

According to other specific embodiments, the antagonist inhibits thebiological function (e.g. signal transduction) of the immune-check pointprotein e.g., as detected by e.g. kinase activity, proliferation assay,cytotoxicity assay or cytokine secretion assay. The reduction may be byat least a 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95% orat least 99% as compared to same in the absence of the antagonist.

Preventing and/or inhibiting the biological function of an immune-checkpoint protein can be effected at the protein level (e.g., antibodies,small molecules, inhibitory peptides, enzymes that cleave thepolypeptide, aptamers and the like) but may also be effected at thegenomic (e.g. homologous recombination and site specific endonucleases)and/or the transcript level using a variety of molecules which interferewith transcription and/or translation (e.g., RNA silencing agents) of aninhibitory immune-check point protein.

Non limiting examples of agents that can function as antagonists aredescribed in details hereinbelow.

Suppressing Biological Function at the Polypeptide Level

According to specific embodiments, the antagonistic agent is anantibody.

According to specific embodiments the antagonistic antibody is capableof specifically binding an inhibitory immune-check point protein.According to specific embodiments, the antagonistic antibodyspecifically binds at least one epitope of an inhibitory immune-checkpoint protein.

As used herein, the term “epitope” refers to any antigenic determinanton an antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or carbohydrate side chains and usuallyhave specific three dimensional structural characteristics, as well asspecific charge characteristics.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, Fv, scFv,dsFv, or single domain molecules such as VH and VL that are capable ofbinding to an epitope of an antigen.

The antibody may be mono-specific (capable of recognizing one epitope orprotein), bi-specific (capable of binding two epitopes or proteins) ormulti-specific (capable of recognizing multiple epitopes or proteins).

Suitable antibody fragments for practicing some embodiments of theinvention include a complementarity-determining region (CDR) of animmunoglobulin light chain (referred to herein as “light chain”), acomplementarity-determining region of an immunoglobulin heavy chain(referred to herein as “heavy chain”), a variable region of a lightchain, a variable region of a heavy chain, a light chain, a heavy chain,an Fd fragment, and antibody fragments comprising essentially wholevariable regions of both light and heavy chains such as an Fv, a singlechain Fv Fv (scFv), a disulfide-stabilized Fv (dsFv), an Fab, an Fab′,and an F(ab′)2.

As used herein, the terms “complementarity-determining region” or “CDR”are used interchangeably to refer to the antigen binding regions foundwithin the variable region of the heavy and light chain polypeptides.Generally, antibodies comprise three CDRs in each of the VH (CDR HI orHI; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LIor LI; CDR L2 or L2; and CDR L3 or L3).

The identity of the amino acid residues in a particular antibody thatmake up a variable region or a CDR can be determined using methods wellknown in the art and include methods such as sequence variability asdefined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service, NIH,Washington D.C.), location of the structural loop regions as defined byChothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), acompromise between Kabat and Chothia using Oxford Molecular's AbMantibody modeling software (now Accelrys®, see, Martin et al., 1989,Proc. Natl Acad Sci USA. 86:9268; and world wide web sitewww(dot)bioinf-org(dot)uk/abs), available complex crystal structures asdefined by the contact definition (see MacCallum et al., J. Mol. Biol.262:732-745, 1996), the “conformational definition” (see, e.g., Makabeet al., Journal of Biological Chemistry, 283:1156-1166, 2008) and IMGT[Lefranc MP, et al. (2003) IMGT unique numbering for immunoglobulin andT cell receptor variable domains and Ig superfamily V-like domains. DevComp Immunol 27: 55-77].

As used herein, the “variable regions” and “CDRs” may refer to variableregions and CDRs defined by any approach known in the art, includingcombinations of approaches.

Functional antibody fragments comprising whole or essentially wholevariable regions of both light and heavy chains are defined as follows:

(i) Fv, defined as a genetically engineered fragment consisting of thevariable region of the light chain (VL) and the variable region of theheavy chain (VH) expressed as two chains;

(ii) single chain Fv (“scFv”), a genetically engineered single chainmolecule including the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule;

(iii) disulfide-stabilized Fv (“dsFv”), a genetically engineeredantibody including the variable region of the light chain and thevariable region of the heavy chain, linked by a genetically engineereddisulfide bond;

(iv) Fab, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme papain to yield the intact lightchain and the Fd fragment of the heavy chain which consists of thevariable and CH1 domains thereof;

(v) Fab′, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme pepsin, followed by reduction(two Fab′ fragments are obtained per antibody molecule);

(vi) F(ab′) 2, a fragment of an antibody molecule containing amonovalent antigen-binding portion of an antibody molecule which can beobtained by treating whole antibody with the enzyme pepsin (i.e., adimer of Fab′ fragments held together by two disulfide bonds); and

(vii) Single domain antibodies or nanobodies are composed of a single VHor VL domains which exhibit sufficient affinity to the antigen.

The antibody may be monoclonal or polyclonal.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to some embodiments of the invention can beprepared by proteolytic hydrolysis of the antibody or by expression inE. coli or mammalian cells (e.g. Chinese hamster ovary cell culture orother protein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by [Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

It will be appreciated that for human therapy or diagnostics, humanizedantibodies are preferably used. Humanized forms of non-human (e.g.,murine) antibodies are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′).sub.2 or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Patent No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10,: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

Once antibodies are obtained, they may be tested for activity, forexample via ELISA.

Another agent which can be used as antagonist with some embodiments ofthe invention is an aptamer. As used herein, the term “aptamer” refersto double stranded or single stranded RNA molecule that binds tospecific molecular target, such as a protein. Various methods are knownin the art which can be used to design protein specific aptamers. Theskilled artisan can employ SELEX (Systematic Evolution of Ligands byExponential Enrichment) for efficient selection as described inStoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering(2007) 24(4):381-403).

Another agent capable of being an antagonist would be any molecule whichinterferes with the immune-check point protein function (e.g. catalyticor interaction) by binding to and/or cleaving the immune-check pointprotein. Such molecules can be, but are not limited to, small molecules,inhibitory peptides, enzymes that cleave the immune-check point protein,adnectins, affibodies, avimers, anticalins, tetranectins, DARPins, andengineered Kunitz-type inhibitors wherein each possibility is a separateembodiment of the invention.

According to a specific embodiment, the antagonist is a small molecule.

According to a specific embodiment, the antagonist is a peptidemolecule.

It will be appreciated that a non-functional analogue of at least acatalytic or binding portion of an inhibitory peptide can be also usedas an antagonist.

Suppressing biological function at the nucleic acid level

Down-regulation at the nucleic acid level is typically effected using anucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimeticsthereof or a combination of same. The nucleic acid agent may be encodedfrom a DNA molecule or provided to the cell per se.

Thus, the antagonist of some embodiments of the invention can be an RNAsilencing agent. As used herein, the phrase “RNA silencing” refers to agroup of regulatory mechanisms [e.g. RNA interference (RNAi),transcriptional gene silencing (TGS), post-transcriptional genesilencing (PTGS), quelling, co-suppression, and translationalrepression] mediated by RNA molecules which result in the inhibition or“silencing” of the expression of a corresponding protein-coding gene.RNA silencing has been observed in many types of organisms, includingplants, animals, and fungi.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of specifically inhibiting or “silencing” the expression of atarget gene. In certain embodiments, the RNA silencing agent is capableof preventing complete processing (e.g., the full translation and/orexpression) of an mRNA molecule through a post-transcriptional silencingmechanism. RNA silencing agents include non-coding RNA molecules, forexample RNA duplexes comprising paired strands, as well as precursorRNAs from which such small non-coding RNAs can be generated. ExemplaryRNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.

In one embodiment, the RNA silencing agent is capable of inducing RNAinterference.

In another embodiment, the RNA silencing agent is capable of mediatingtranslational repression.

According to an embodiment of the invention, the RNA silencing agent isspecific to the target RNA (i.e. an immune-check point e.g. PD-1, PDL-1,CTLA-4, LAG-3, TIM-3, KIR and IDO) and does not cross inhibit or silenceother targets or a splice variant which exhibits 99% or less globalhomology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% globalhomology to the target gene; as determined by PCR, Western blot,Immunohistochemistry and/or flow cytometry.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs).

Following is a detailed description on RNA silencing agents that can beused according to specific embodiments of the present invention.

DsRNA, siRNA and shRNA - The presence of long dsRNAs in cells stimulatesthe activity of a ribonuclease III enzyme referred to as dicer. Dicer isinvolved in the processing of the dsRNA into short pieces of dsRNA knownas short interfering RNAs (siRNAs). Short interfering RNAs derived fromdicer activity are typically about 21 to about 23 nucleotides in lengthand comprise about 19 base pair duplexes.

The RNA₁ response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex.

Accordingly, some embodiments of the invention contemplate use of dsRNAto downregulate protein expression from mRNA.

According to one embodiment dsRNA longer than 30 bp are used. Variousstudies demonstrate that long dsRNAs can be used to silence geneexpression without inducing the stress response or causing significantoff-target effects—see for example [Strat et al., Nucleic AcidsResearch, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004;13:115-125; Diallo M., et al., Oligonucleotides.2003;13:381-392; Paddison P.J., et al., Proc. Natl Acad. Sci. USA.2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004;573:127-134].

According to some embodiments of the invention, dsRNA is provided incells where the interferon pathway is not activated, see for exampleBilly et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al.,Oligonucleotides, October 1, 2003, 13(5): 381-392.doi:10.1089/154545703322617069.

According to an embodiment of the invention, the long dsRNA arespecifically designed not to induce the interferon and PKR pathways fordown-regulating gene expression. For example, Shinagwa and Ishii [Genes& Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP,to express long double-strand RNA from an RNA polymerase II (Pol II)promoter. Because the transcripts from pDECAP lack both the 5′-capstructure and the 3′-poly(A) tail that facilitate ds-RNA export to thecytoplasm, long ds-RNA from pDECAP does not induce the interferonresponse.

Another method of evading the interferon and PKR pathways in mammaliansystems is by introduction of small inhibitory RNAs (siRNAs) either viatransfection or endogenous expression.

The term “siRNA” refers to small inhibitory RNA duplexes (generallybetween 18-30 base pairs) that induce the RNA interference (RNAi)pathway.

Typically, siRNAs are chemically synthesized as 2lmers with a central 19bp duplex region and symmetric 2-base 3′-overhangs on the termini,although it has been recently described that chemically synthesized RNAduplexes of 25-30 base length can have as much as a 100-fold increase inpotency compared with 2lmers at the same location. The observedincreased potency obtained using longer RNAs in triggering RNA₁ issuggested to result from providing Dicer with a substrate (27mer)instead of a product (21mer) and that this improves the rate orefficiency of entry of the siRNA duplex into RISC.

It has been found that position of the 3′-overhang influences potency ofan siRNA and asymmetric duplexes having a 3′-overhang on the antisensestrand are generally more potent than those with the 3′-overhang on thesense strand (Rose et al., 2005). This can be attributed to asymmetricalstrand loading into RISC, as the opposite efficacy patterns are observedwhen targeting the antisense transcript.

The strands of a double-stranded interfering RNA (e.g., an siRNA) may beconnected to form a hairpin or stem-loop structure (e.g., an shRNA).Thus, as mentioned, the RNA silencing agent of some embodiments of theinvention may also be a short hairpin RNA (shRNA).

The term “shRNA”, as used herein, refers to an RNA agent having astem-loop structure, comprising a first and second region ofcomplementary sequence, the degree of complementarity and orientation ofthe regions being sufficient such that base pairing occurs between theregions, the first and second regions being joined by a loop region, theloop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region. The number of nucleotides inthe loop is a number between and including 3 to 23, or 5 to 15, or 7 to13, or 4 to 9, or 9 to 11.

Some of the nucleotides in the loop can be involved in base-pairinteractions with other nucleotides in the loop. Examples ofoligonucleotide sequences that can be used to form the loop include5′-CAAGAGA-3′ and 5′ -UUACAA-3′ (International Patent Application Nos.WO2013126963 and WO2014107763). It will be recognized by one of skill inthe art that the resulting single chain oligonucleotide forms astem-loop or hairpin structure comprising a double-stranded regioncapable of interacting with the RNA₁ machinery.

Synthesis of RNA silencing agents suitable for use with some embodimentsof the invention can be effected as follows. First, the inhibitory-checkpoint mRNA sequence is scanned downstream of the AUG start codon for AAdinucleotide sequences. Occurrence of each AA and the 3′ adjacent 19nucleotides is recorded as potential siRNA target sites. Preferably,siRNA target sites are selected from the open reading frame, asuntranslated regions (UTRs) are richer in regulatory protein bindingsites. UTR-binding proteins and/or translation initiation complexes mayinterfere with binding of the siRNA endonuclease complex [TuschlChemBiochem. 2:239-245]. It will be appreciated though, that siRNAsdirected at untranslated regions may also be effective, as demonstratedfor GAPDH wherein siRNA directed at the 5′ UTR mediated about 90%decrease in cellular GAPDH mRNA and completely abolished protein level(www(dot)ambion(dot)com/techlib/tn/91/912(dot)html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/). Putative target siteswhich exhibit significant homology to other coding sequences arefiltered out.

Qualifying target sequences are selected as template for siRNAsynthesis.

Preferred sequences are those including low G/C content as these haveproven to be more effective in mediating gene silencing as compared tothose with G/C content higher than 55%. Several target sites arepreferably selected along the length of the target gene for evaluation.For better evaluation of the selected siRNAs, a negative control ispreferably used in conjunction. Negative control siRNA preferablyinclude the same nucleotide composition as the siRNAs but lacksignificant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

For example, suitable siRNAs directed against PDL-1 can be obtained fromThermo Fisher Scientific (St Leon-Rot, Germany) and Invitrogen(Carlsbad, CA, USA); siRNAs directed against PDL-2 can be obtained fromInvitrogen (Carlsbad, CA, USA); siRNA directed against IDO can be asdescribed in Zheng et al. The journal of Immunology (2006) 177(8):5639-5646.

It will be appreciated that, and as mentioned hereinabove, the RNAsilencing agent of some embodiments of the invention need not be limitedto those molecules containing only RNA, but further encompasseschemically-modified nucleotides and non-nucleotides.

According to another embodiment the RNA silencing agent may be a miRNA.

The term “microRNA”, “miRNA”, and “miR” are synonymous and refer to acollection of non-coding single-stranded RNA molecules of about 19-28nucleotides in length, which regulate gene expression. miRNAs are foundin a wide range of organisms (viruses.fwdarw.humans) and have been shownto play a role in development, homeostasis, and disease etiology.

Below is a brief description of the mechanism of miRNA activity.

Genes coding for miRNAs are transcribed leading to production of anmiRNA precursor known as the pri-miRNA. The pri-miRNA may form a hairpinwith a stem and loop.

The hairpin structure of the pri-miRNA is recognized by Drosha, which isan RNase III endonuclease. Drosha typically recognizes terminal loops inthe pri-miRNA and cleaves the pri-miRNA with a staggered cut typical ofRNase III endonucleases yielding a pre-miRNA stem loop with a 5′phosphate and ˜2 nucleotide 3′ overhang. The pre-miRNA is then activelytransported from the nucleus to the cytoplasm by Ran-GTP and the exportreceptor Ex-portin-5.

The double-stranded stem or the 5′ phosphate and 3′ overhang at the baseof the stem loop of the pre-miRNA is then recognized by Dicer, which isalso an RNase III endonuclease. Dicer then cleaves off the terminal looptwo helical turns away from the base of the stem loop leaving anadditional 5′ phosphate and ˜2 nucleotide 3′ overhang. The resultingsiRNA-like duplex, which may comprise mismatches, comprises the maturemiRNA and a similar-sized fragment known as the miRNA*. miRNA* sequencesmay be found in libraries of cloned miRNAs but typically at lowerfrequency than the miRNAs.

Although initially present as a double-stranded species with miRNA*, themiRNA eventually becomes incorporated as a single-stranded RNA into aribonucleoprotein complex known as the RNA-induced silencing complex(RISC) while the miRNA* is removed and degraded.

The RISC identifies target nucleic acids based on high levels ofcomplementarity between the miRNA and the mRNA, especially bynucleotides 2-7 of the miRNA.

A number of studies have looked at the base-pairing requirement betweenmiRNA and its mRNA target for achieving efficient inhibition oftranslation (reviewed by Bartel 2004, Cell 116-281). In mammalian cells,the first 8 nucleotides of the miRNA may be important (Doench & Sharp2004 GenesDev 2004-504). However, other parts of the microRNA may alsoparticipate in mRNA binding. Moreover, sufficient base pairing at the 3′can compensate for insufficient pairing at the 5′ (Brennecke et al.,2005 PLoS 3-e85). Computation studies, analyzing miRNA binding on wholegenomes have suggested a specific role for bases 2-7 at the 5′ of themiRNA in target binding but the role of the first nucleotide, foundusually to be “A” was also recognized (Lewis et at 2005 Cell 120-15).Similarly, nucleotides 1-7 or 2-8 were used to identify and validatetargets by Krek et al. (2005, Nat Genet 37-495).

The target sites in the mRNA may be in the 5′ UTR, the 3′ UTR or in thecoding region.

miRNAs may direct the RISC to downregulate gene expression by either oftwo mechanisms: mRNA cleavage or translational repression. The miRNA mayspecify cleavage of the mRNA if the mRNA has a certain degree ofcomplementarity to the miRNA. When a miRNA guides cleavage, the cut istypically between the nucleotides pairing to residues 10 and 11 of themiRNA. Alternatively, the miRNA may repress translation if the miRNAdoes not have the requisite degree of complementarity to the miRNA.

It will be appreciated from the description provided herein above thatcontacting cells with a miRNA may be effected by transfecting/loadingthe cells with e.g. the mature double stranded miRNA, the pre-miRNA orthe pri-miRNA.

The pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70nucleotides.

The pri-miRNA sequence may comprise from 45-30,000, 50-25,000,100-20,000, 1,000-1,500 or 80-100 nucleotides.

Antisense—Antisense is a single stranded RNA designed to prevent orinhibit expression of a gene by specifically hybridizing to its mRNA.Downregulation of an immune-check point can be effected using anantisense polynucleotide capable of specifically hybridizing with anmRNA transcript encoding the immune-check point protein.

Design of antisense molecules which can be used to efficientlydownregulate an immune-check point must be effected while consideringtwo aspects important to the antisense approach. The first aspect isdelivery of the oligonucleotide into the cytoplasm of the appropriatecells, while the second aspect is design of an oligonucleotide whichspecifically binds the designated mRNA within cells in a way whichinhibits translation thereof.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Jääskeläinen et al. Cell Mol Biol Lett. (2002)7(2):236-7; Gait, Cell Mol Life Sci. (2003) 60(5):844-53; Martino et al.J Biomed Biotechnol. (2009) 2009:410260; Grijalvo et al. Expert OpinTher Pat. (2014) 24(7):801-19; Falzarano et al., Nucleic Acid Ther.(2014) 24(1):87-100; Shilakari et al. Biomed Res Int. (2014) 2014:526391; Prakash et al. Nucleic Acids Res. (2014) 42(13):8796-807 andAsseline et al. J Gene Med. (2014) 16(7-8):157-65].

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)]. Such algorithms have been successfully used to implement anantisense approach in cells.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished [Matveeva et al., Nature Biotechnology 16: 1374 - 1375(1998)].

Thus, the generation of highly accurate antisense design algorithms anda wide variety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

Nucleic acid agents can also operate at the DNA level as summarizedinfra.

Suppressing the biological function of an immune-check point can also beachieved by inactivating the gene (e.g., PD-1, PDL-1, CTLA-4, LAG-3,TIM-3, KIR and IDO) via introducing targeted mutations involving loss-offunction alterations (e.g. point mutations, deletions and insertions) inthe gene structure.

As used herein, the phrase “loss-of-function alterations” refers to anymutation in the DNA sequence of a gene which results in downregulationof the expression level and/or activity of the expressed product, i.e.,the mRNA transcript and/or the translated protein. Non-limiting examplesof such loss-of-function alterations include a missense mutation, i.e.,a mutation which changes an amino acid residue in the protein withanother amino acid residue and thereby abolishes the enzymatic activityof the protein; a nonsense mutation, i.e., a mutation which introduces astop codon in a protein, e.g., an early stop codon which results in ashorter protein devoid of the enzymatic activity; a frame-shiftmutation, i.e., a mutation, usually, deletion or insertion of nucleicacid(s) which changes the reading frame of the protein, and may resultin an early termination by introducing a stop codon into a reading frame(e.g., a truncated protein, devoid of the enzymatic activity), or in alonger amino acid sequence (e.g., a readthrough protein) which affectsthe secondary or tertiary structure of the protein and results in anon-functional protein, devoid of the enzymatic activity of thenon-mutated polypeptide; a readthrough mutation due to a frame-shiftmutation or a modified stop codon mutation (i.e., when the stop codon ismutated into an amino acid codon), with an abolished enzymatic activity;a promoter mutation, i.e., a mutation in a promoter sequence, usually 5′to the transcription start site of a gene, which results indown-regulation of a specific gene product; a regulatory mutation, i.e.,a mutation in a region upstream or downstream, or within a gene, whichaffects the expression of the gene product; a deletion mutation, i.e., amutation which deletes coding nucleic acids in a gene sequence and whichmay result in a frame-shift mutation or an in-frame mutation (within thecoding sequence, deletion of one or more amino acid codons); aninsertion mutation, i.e., a mutation which inserts coding or non-codingnucleic acids into a gene sequence, and which may result in aframe-shift mutation or an in-frame insertion of one or more amino acidcodons; an inversion, i.e., a mutation which results in an invertedcoding or non-coding sequence; a splice mutation i.e., a mutation whichresults in abnormal splicing or poor splicing; and a duplicationmutation, i.e., a mutation which results in a duplicated coding ornon-coding sequence, which can be in-frame or can cause a frame-shift.

According to specific embodiments los-of-function alteration of a genemay comprise at least one allele of the gene.

The term “allele” as used herein, refers to any of one or morealternative forms of a gene locus, all of which alleles relate to atrait or characteristic. In a diploid cell or organism, the two allelesof a given gene occupy corresponding loci on a pair of homologouschromosomes.

According to other specific embodiments loss-of-function alteration of agene comprises both alleles of the gene.

Methods of introducing nucleic acid alterations to a gene of interestare well known in the art [see for example Menke D. Genesis (2013) 51:—618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc NatlAcad Sci USA (2008) 105:5809-5814; International Patent Application Nos.WO2014085593, WO2009071334 and WO2011146121; U.S. Pat. Nos. 8,771,945,8,586,526, 6,774,279 and U.S. Patent Application Publication Nos.20030232410, 20050026157, US20060014264; the contents of which areincorporated by reference in their entireties] and include targetedhomologous recombination (e.g. “Hit and run”, “double-replacement”),site specific recombinases (e.g. the Cre recombinase and the Flprecombinase), PB transposases (e.g. Sleeping Beauty, piggyBac, To12 orFrog Prince), genome editing by engineered nucleases (e.g.meganucleases, Zinc finger nucleases (ZFNs), transcription-activatorlike effector nucleases (TALENs) and CRISPR/Cas system) and genomeediting using recombinant adeno-associated virus (rAAV) platform. Agentsfor introducing nucleic acid alterations to a gene of interest can bedesigned publically available sources or obtained commercially fromTransposagen, Addgene and Sangamo Biosciences.

Methods for qualifying efficacy and detecting sequence alteration arewell known in the art and include, but not limited to, DNA sequencing,electrophoresis, an enzyme-based mismatch detection assay and ahybridization assay such as PCR, RT-PCR, RNase protection, in-situhybridization, primer extension, Southern blot, Northern Blot and dotblot analysis.

Sequence alterations in a specific gene can also be determined at theprotein level using e.g. chromatography, electrophoretic methods,immunodetection assays such as ELISA and western blot analysis andimmunohistochemistry.

As mentioned, depending on the immune-check point protein (i.e.co-stimulatory or inhibitory) the immune-check point regulator can be anagonist or antagonist. Thus, according to specific embodiments, theimmune-check point regulator is an agonist.

As used herein the term “agonist” refers to a molecule that inducesand/or increases the biological function and/or expression of animmune-check point protein.

According to specific embodiments, the agonist induces and/or increasesthe co-stimulatory effect of an immune-check point protein on an immunecell (e.g. T cells).

According to specific embodiments, the agonist induces and/or increasessignaling to an immune cell (e.g. T cell) by an immune-check pointprotein.

The agonist can be a naturally occurring activator or a functionalderivative thereof; or non-naturally occurring activator.

According to specific embodiments, the agonist is a full agonist, thatis, the effect of the agonist is equivalent to the effect of thenaturally occurring activator (i.e. ligand).

According to other specific embodiments, the agonist is a partialagonist, that is, the effect of the agonist is lower than the maximaleffect of the naturally occurring activator (i.e. ligand). The effect ofthe agonist may be lower by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40% at least 50%, at least 60%, at least 70%, atleast 80% or at least 90% as compared to the maximal effect of thenaturally occurring activator.

According to yet other specific embodiments, the agonist is a superagonist, that is, the effect of the agonist is higher than the maximaleffect of the naturally occurring activator (i.e. ligand). The effect ofthe agonist may be higher by at least 5%, at least 10% , at least 20%,at least 30%, at least 40% at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 2 fold, at least 4 fold, at least 5fold or at least 10 fold as compared to the maximal effect of thenaturally occurring activator.

According to specific embodiments, the agonist induces completeactivation the biological function (e.g. signal transduction) of theimmune-check point protein.

According to other specific embodiments, the agonist increases thebiological function (e.g. signal transduction) of the immune-check pointprotein e.g., as detected by e.g. kinase activity, proliferation assay,cytotoxicity assay or cytokine secretion assay. The increase may be byat least a 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95% orat least 99% as compared to same in the absence of the agonist.

According to specific embodiments, the agonist binds directly theimmune-check point protein.

According to other specific embodiments, the agonist indirectly bindsthe immune-check point protein by acting through an intermediarymolecule, for example the agonist binds to or modulates a molecule thatin turn binds to or modulates the immune-check point protein.

Activating and/or increasing the biological function of an immune-checkpoint protein can be effected at the protein level (e.g., antibodies,small molecules, peptides and the like) but may also be effected at thegenomic level (e.g., activation of transcription via promoters,enhancers, regulatory elements) and/or the transcript level using avariety of molecules which promote transcription and/or translation(e.g., correct splicing, polyadenylation, activation of translation) ofa co-stimulatory immune-check point protein.

Non limiting examples of agents that can function as agonists aredescribed in details hereinbelow.

Activating and/or Increasing Biological Function at the PolypeptideLevel

According to specific embodiments, the agonist is the naturallyoccurring activator or a functional derivative or variant thereof whichretain the ability to specifically bind to the immune-check pointprotein.

It will be appreciated that a functional analogue of at least acatalytic or binding portion of a co-stimulatory peptide can be alsoused as an agonist. Thus, according to specific embodiments, the agonistis an exogenous polypeptide including at least a functional portion(e.g. catalytic or interaction) of the co-stimulatory immune-check pointprotein. Thus, for example, the polypeptide can be a ligand capable ofbinding and activating the co-stimulatory immune-check point proteinreceptor.

According to specific embodiments, the agonist is an antibody.

According to specific embodiments the agonistic antibody is capable ofspecifically binding a co-stimulatory immune-check point protein.According to specific embodiments, the agonistic antibody specificallybinds at least one epitope of a co-stimulatory immune-check pointprotein. A detailed description on antibodies that can be used accordingto specific embodiments of the present invention is providedhereinabove.

Another agent capable of being an agonist would be a molecule whichpromotes and/or increases the co-stimulatory immune-check point proteinfunction (e.g. catalytic or interaction) by binding to the immune-checkpoint protein or an intermediate thereof. Such molecules can be, but arenot limited to, small molecules, peptides, aptamers, adnectins,affibodies, avimers, anticalins, tetranectins and DARPins, wherein eachpossibility is a separate embodiment of the invention.

According to specific embodiments, the agonist is a small molecule.

According to specific embodiments, the agonist is a peptide.

Activating and/or Increasing Biological Function at the Nucleic AcidLevel

An agonist can also be a molecule which is capable of increasing thetranscription and/or translation of an endogenous DNA or mRNA encodingthe co-stimulatory immune-check point protein and thus increasingendogenous co-stimulatory immune-check point protein activity.

Another agonistic agent may be an exogenous polynucleotide (DNA or RNA)sequence designed and constructed to express at least a functionalportion of the co-stimulatory immune-check point protein.

Several co-stimulatory immune-check points have been cloned from human,rat and mouse sources. Thus, coding sequences information is availablefrom several databases including the GenBank database available throughwww(dot)ncbi(dot)nlm(dot)nih(dot)gov/.

To express an exogenous co-stimulatory immune-check point protein inmammalian cells, a polynucleotide sequence encoding a specificco-stimulatory immune-check point protein or a homologue thereof whichexhibit the desired activity is preferably ligated into a nucleic acidconstruct suitable for mammalian cell expression. Such a nucleic acidconstruct includes a promoter sequence for directing transcription ofthe polynucleotide sequence in the cell in a constitutive [e.g.cytomegalovirus (CMV) and Rous sarcoma virus (RSV)] or inducible (e.g.the tetracycline-inducible promoter) manner.

According to specific embodiments, the promoter utilized by the nucleicacid construct of some embodiments of the invention is active in aspecific cell population. Examples of cell type-specific and/ortissue-specific promoters include promoters such as, but not limited tolymphoid specific promoters [Calame et al., (1988) Adv. Immunol.43:235-275]; in particular promoters of T-cell receptors [Winoto et al.,(1989) EMBO J. 8:729-733] and immunoglobulins [Banerji et al. (1983)Cell 33729-740].

The nucleic acid construct (also referred to herein as an “expressionvector”) of some embodiments of the invention includes additionalsequences which render this vector suitable for replication andintegration in prokaryotes, eukaryotes, or preferably both (e.g.,shuttle vectors). In addition, a typical cloning vectors may alsocontain a transcription and translation initiation sequence,transcription and translation terminator and a polyadenylation signal.By way of example, such constructs will typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof. The construct may alsoinclude an enhancer element which can stimulate transcription up to1,000 fold from linked homologous or heterologous promoters. The vectormay or may not include a eukaryotic replicon.

The nucleic acid construct of some embodiments of the invention can alsoinclude a signal sequence for secretion of the peptide from a host cellin which it is placed. Preferably the signal sequence for this purposeis a mammalian signal sequence or the signal sequence of the polypeptidevariants of some embodiments of the invention.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of a co-stimulatory immune-check pointmRNA translation. Two distinct sequence elements are required foraccurate and efficient polyadenylation: GU or U rich sequences locateddownstream from the polyadenylation site and a highly conserved sequenceof six nucleotides, AAUAAA, located 11-30 nucleotides upstream.Termination and polyadenylation signals that are suitable for someembodiments of the invention include those derived from SV40.

The expression vector of some embodiments of the invention can furtherinclude additional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

Other than containing the necessary elements for the transcription andtranslation of the inserted coding sequence, the expression construct ofsome embodiments of the invention can also include sequences engineeredto enhance stability, production, or yield of the expressed peptide.

It will be appreciated that the individual elements comprised in theexpression vector can be arranged in a variety of configurations.

The type of vector used by some embodiments of the invention will dependon the cell type transformed. The ability to select suitable vectorsaccording to the cell type transformed is well within the capabilitiesof the ordinary skilled artisan and as such no general description ofselection consideration is provided herein.

Recombinant viral vectors are useful for in vivo expression of animmune-check point protein since they offer advantages such as lateralinfection and targeting specificity. Viral vectors can also be producedthat are unable to spread laterally.

Various methods can be used to introduce the expression vector of someembodiments of the invention into cells. Such methods are generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986]. Currently preferred in vivonucleic acid transfer techniques include transfection with viral ornon-viral constructs, such as adenovirus, lentivirus, Herpes simplex Ivirus, or adeno-associated virus (AAV) and lipid-based systems. Usefullipids for lipid-mediated transfer of the gene are, for example, DOTMA,DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65(1996)]. The most preferred constructs for use in gene therapy areviruses, most preferably adenoviruses, AAV, lentiviruses, orretroviruses. Other vectors can be used that are non-viral, such ascationic lipids, polylysine, and dendrimers.

As mentioned, the CXCR4 antagonistic peptides of the present inventionmay be used in combination with an immune-check point regulator for thetreatment of cancer. According to specific embodiments, wherein theimmune-check protein is an inhibitory protein, the CXCR4 antagonisticpeptides of the present invention are administered in combination withan immune-check point antagonist. Following is a list of combinationsthat may be used in accordance with the present teachings.

Thus, according to an aspect of the present invention there is provideda method of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aPD1 antagonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aPD1 antagonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a PD1 antagonist; and a pharmaceuticallyacceptable carrier or diluent.

PD1 (Programmed Death 1, also known as CD279) is a 55 kDa type Itransmembrane protein that is part of the Ig gene superfamily, which isexpressed on the surface of several immune cells such as activated Tcells, B cells, NK cells and myeloid cells. PD-1 contains a membraneproximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membranedistal tyrosine-based switch motif (ITSM).

The presence of an ITIM on PD-1 indicates that this molecule functionsto attenuate antigen receptor signaling by recruitment of cytoplasmicphosphatases.

According to a specific embodiment, the PD1 protein refers to the humanprotein, such as provided in the following GenBank Number NP_005009. Twoligands for PD-1 have been identified, PD-L1 and PD-L2 (also known asB7-DC).

According to a specific embodiment, the PD-L 1 protein refers to thehuman protein, such as provided in the following GenBank NumberNP_001254635 and NP_054862. According to a specific embodiment, thePD-L2 protein refers to the human protein, such as provided in thefollowing GenBank Number NP_079515.

PD1 pattern of expression and function is dependent on the cell type.PD1 expression is induced following effector T cells activation. Uponligand binding, PD1 inhibits kinases that are involved in T cellactivation through e.g. the phosphatase SHP2, thereby transmits aninhibitory signal. Conversely, PD1 is highly expressed on regulatory Tcells, where it may enhance their proliferation upon ligand binding.PD-1 is also induced on other activated non-T lymphocyte subsets,including B cells and NK cells, where upon ligand binding it transmitsan inhibitory signal which limits their antibody production and lyticactivity, respectively [Pardon (2012) Nature Reviews Cancer 12,252-264].

Thus, PD1 blockade may enhance the activity of effector T cells, NKcells and antibody production in tissues and in the tumormicroenvironment. Because many tumors are highly infiltrated withregulatory T cells that probably further suppress effector immuneresponses, blockade of the PD1 pathway may also enhance antitumor immuneresponses by diminishing the number and/or suppressive activity ofintra-tumoral regulatory T cells.

As used herein, the term “PD1 antagonist” refers to an antagonisticagent that prevents and/or inhibits the biological function and/orexpression of PD1.

According to specific embodiments, the PD1 antagonist prevents and/orinhibits signaling to an immune cell (e.g. T cells, B cells, NK cells)by PD1; thereby suppresses PD1 immune-suppressive activity.

According to specific embodiments, the PD1 antagonist promotes immuneresponse of an effector T cell following TCR activating signal.

According to specific embodiments, the PD1 antagonist of the presentinvention binds directly PD1 and/or binds a ligand of PD1 and interfereswith and/or inhibits the binding of the ligands to PD1.

According to other specific embodiments, the PD1 antagonist indirectlybinds PD1 by acting through an intermediary molecule, for example theantagonist binds to or modulates a molecule that in turn binds to ormodulates PD1.

According to specific embodiments, the PD1 antagonist binds PD1.According to other specific embodiments, the PD1 antagonist binds atleast one of the PD-1 ligands (e.g. PDL-1 or PDL-2), as furtherdescribed hereinbelow.

In certain embodiments, the PD1 antagonist exhibits one or moredesirable functional properties, such as high affinity binding to PD1,e.g., binding to human PD1 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰M or less; lack of significant cross-reactivity to other immune-checkpoint proteins, e.g., CD28, CTLA-4 and ICOS; the ability to stimulate Tcell proliferation; the ability to increase IFN-y and/or IL-2 secretion;the ability to inhibit binding of one or more PD1 ligands (e.g., PD-L1and/or PD-L2) to PD1; the ability to stimulate antigen-specific memoryresponses; the ability to stimulate antibody responses and/or theability to inhibit growth of tumor cells.

According to a specific embodiment, the PD1 antagonist is an antibody.

According to specific embodiments, the PD1 antagonist is an anti-PD1antibody. Anti-PD1 antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognized anti-PD1antibodies can be used. Examples of anti-PD1 antibodies are disclosedfor example in Topalian, et al. NEJM 2012, U.S. Pat. Nos. 7,488,802;8,008,449; 8,609,089; 6,808,710; 7,521,051; and 8168757, U.S. PatentApplication Publication Nos. 20140227262; 20100151492; 20060210567; and20060034826 and International Patent Application Publication Nos.WO2008156712; WO2010089411; WO2010036959; WO2011159877; WO2013/019906;WO2014159562; WO2011109789; WO01/14557; WO2004/004771; andWO2004/056875, which are hereby incorporated by reference in theirentirety.

Specific anti-PD1 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

Nivolumab (also known as MDX1106, BMS-936558, ONO-4538), marketed by BMYas Opdivo, a fully human IgG4 antibody with the structure described inWHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013) that binds toand blocks the activation of PD-1 by its ligands PD-L1 and PD-L2;

Pembrolizumab (also known as MK-3475, Keytruda, SCH 900475, produced byMerck), a humanized monoclonal IgG4 antibody with the structuredescribed in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013)that binds to and blocks the activation of PD1 by its ligands;

Pidilizumab (also known as CT-011, hBAT, hBAT-1, produced by CureTech),a humanized monoclonal IgG1 antibody that binds PD-1;

AMP-514 (also known as MEDI-0680, produced by AZY and MedImmune), ahumanized monoclonal IgG4 antibody that binds PD-1.

Humanized antibodies h409A11, h409A16 and h409A17, which are describedin PCT Patent Application No. WO2008/156712.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to PD1.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on PD1 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

According to a specific embodiment, the PD1 antagonist is an anti-PDL-1antibody, as further described hereinbelow.

According to a specific embodiment, the PD1 antagonist is an anti-PDL-2antibody.

Anti-PDL-2 antibodies suitable for use in the invention can be generatedusing methods well known in the art especially in light of the detaileddescription hereinabove. Alternatively, art recognized anti-PDL-2antibodies can be used. Examples of anti-PD1 antibodies are disclosedfor example in International Application Publication Nos. WO03/042402and WO02/00730, which are hereby incorporated by reference in theirentirety.

Other PD1 antagonists include an immunoadhesin that specifically bindsto PD-1 or any one of its ligands, such as a fusion protein containingthe extracellular or PD1 binding portion of PD-L1 or PD-L2 fused to aconstant region such as an Fc region of an immunoglobulin molecule.Examples of immunoadhesin molecules that specifically bind to PD1 aredescribed in International Patent Application Publication Nos.WO2010/027827 and WO2011/066342, which are hereby incorporated byreference in their entirety.

A specific fusion protein that can be used according to some embodimentsof the present invention is AMP-224 (also known as B7-DCIg, produced byAZY and GSK), an engineered recombinant fusion protein comprised ofhuman PD-L2 and the Fc domain of human IgG1.

Other PD1 antagonists that can be used according to some embodiments ofthe present invention including nucleotides, expression vectors, smallmolecules, peptides, fusion proteins and fragments targeting PD1, PDL-1or PDL-2, non-functional PD1, soluble PD1 or fragments thereof that bindto PD1 ligands and prevent binding to the endogenous PD1 receptor, aredisclosed for examples in U.S. Pat. Nos. 8,609,089 and 6,808,710, U.S.Patent Application Publication Nos. 20140227262; 20100151492;20040137577; 20030232323; 20030044768; 20030039653; 20020164600;20020110836; 20020107363; 20020106730; 20090305950 and 20140271677,International Patent Application Publication Nos. WO03042402;WO2010036959; WO2011066342; WO2011082400; WO2011161699; WO2014012479;WO2011109789; and WO2013132317, which are hereby incorporated byreference in their entirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aPD-L1 antagonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aPD-L1 antagonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a PD-L1 antagonist; and a pharmaceuticallyacceptable carrier or diluent.

PDL-1 (also known as B7-H1) is a B7 homolog that binds PD1 and B7.1(CD80). PDL-1 is constitutively expressed on T cells, B cells, dendriticcells, macrophages, mesenchymal stem cells, bone marrow-derived mastcells and also on a wide range of non-hematopoietic cells (e.g., cornea,lung, vascular epithelium, liver non-parenchymal cells, pancreaticislets, placental synctiotrophoblasts, keratinocytes, etc.) and isupregulated on a number of cell types after activation [Yamazaki et al.,J. Immunol. 169: 5538-45 (2002); Keir et al., Annu. Rev. Immunol. 26:677-704 (2008)]. In addition, many cancerous cells such as, but notlimited to, melanoma, ovarian and lung cancerous cells express PDL-1[Pardon (2012) Nature Reviews Cancer 12, 252-264]. According to aspecific embodiment the PDL-1 protein refers to the human protein, suchas provided in the following GenBank accession Numbers NP_054862 andNP_054862.

Binding of PDL-1 to PD1 or B7.1 has been shown to downregulate T cellactivation [Butte et al. (2007) Immunity 27: 111-22]. It has also beenshown that the interaction between PD-1 and PD-L1 results in a decreasein tumor infiltrating lymphocytes, a decrease in T-cell receptormediated proliferation, and immune evasion by the cancerous cells (Donget al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol.Immunother. 54:307- 314; Konishi et al. (2004) Clin. Cancer Res.10:5094-100).

As used herein, the term “PDL-1 antagonist” refers to an antagonisticagent that prevents and/or inhibits the biological function and/orexpression of PDL-1.

According to specific embodiments, the PDL-1 antagonist prevents and/orinhibits signaling to an immune cell (e.g. T cells, B cells, NK cells)by the interaction of PDL-1 with at least one of its binding partners(e.g. PD-1 and B7.1); thereby suppresses PDL- 1 immune-suppressiveactivity.

According to specific embodiments, the PDL-1 antagonist binds to orinhibits PD-L1 from binding and/or activating its binding partner.

According to specific embodiments, the PDL-1 antagonist binds PDL-1 andinterferes with and/or inhibits the binding of PDL-1 to PD1 and/or B7.1.

According to specific embodiments, the PDL-1 antagonist binds PDL-1 andprevents and/or inhibits PD1 and/or B7.1 activation by the PDL-1.

According to other specific embodiments, the PDL-1 antagonist indirectlybinds PDL-1 by acting through an intermediary molecule, for example theantagonist binds to or modulates a molecule that in turn binds to ormodulates PDL-1.

According to specific embodiments, the PDL-1 antagonist exhibits one ormore desirable functional properties, such as high affinity binding toPDL-1, e.g., binding to human PDL-1 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹M, 10⁻¹⁰ M or less; lack of significant cross-reactivity to otherimmune-check point proteins; the ability to stimulate T cellproliferation; the ability to increase IFN-y and/or IL-2 secretion; theability to stimulate antibody responses and/or the ability to inhibitgrowth of tumor cells.

According to a specific embodiment, the PDL-1 antagonist is ananti-PDL-1 antibody.

Anti-PDL-1 antibodies suitable for use in the invention can be generatedusing methods well known in the art especially in light of the detaileddescription hereinabove. Alternatively, art recognized anti-PDL-1antibodies can be used. Examples of anti-PDL-1 antibodies are disclosedfor example in Brahmer, et al. NEJM 2012, U.S. Pat. Nos. 7,943,743;8,217,149; 8,741,295; 8,552,154; and 8,383,796, U.S. Patent ApplicationPublication Nos. 20140227262; and 20030232323, International PatentApplication Publication Nos. WO2014066834; WO2010036959; WO2011066342;WO2013/019906, WO2010/077634; WO2002079499; WO2003042402, WO2002086083;WO2001039722; WO2007005874 WO2011109789; and WO2007005874, which arehereby incorporated by reference in their entirety.

Specific anti-PDL-1 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

MPDL3280A (also known as RG7446, produced by Roche/Genentech), a human,Fc optimized, monoclonal antibody that binds PD-L1 and prevents itsbinding to and activation of PD1 and B7.1. This antibody contains anengineered Fc domain designed to optimize efficacy and safety byminimizing antibody-dependent cellular cytotoxicity (ADCC);

BMS-936559 (produced by BMS), a fully human IgG4 anti-PD-L1 monoclonalantibody that inhibits the binding of the PD-L1 to both PD-1 and B7.1;

MEDI4736 (also known as Anti-B7-H1, produced by Astra7eneca), amonoclonal antibody that binds PDL-1;

Avelumab (also known as MSB0010718C, produced by Merck KGaA), a fullyhuman anti-PD-L1 IgG1 monoclonal antibody; and

Monoclonal antibodies 12A4, 3G10, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6,12B7, and 13G4 described in International Application Publication No.WO2007/005874 and U.S. Pat. No. 7,943,743.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to PDL-1.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on PDL-1 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

Other PDL-1 antagonists that can be used according to some embodimentsof the present invention include nucleotides, expression vectors, smallmolecules, peptides, immunoadhesins, fusion proteins and fragmentstargeting PDL-1, non-functional PDL-1 or fragments thereof that bind tobut do not promote signaling by PD1, are disclosed for examples in U.S.Pat. Nos. 6,808,710; U.S. Patent Application Publication Nos.20140227262; 20030232323; 20030039653; and 20140271677; InternationalPatent Application Publication Nos. WO2011066342; WO2013/019906; andWO2011109789, which are hereby incorporated by reference in theirentirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCTLA-4 antagonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof; and aCTLA-4 antagonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof; and a CTLA-4 antagonist; and a pharmaceuticallyacceptable carrier or diluent.

CTLA4 is a member of the immunoglobulin superfamily, which is expressedon the surface of T cells (e.g. CD4+helper T cells) and transmits aninhibitory signal to T cells upon ligand binding. According to aspecific embodiment the CTLA-4 protein refers to the human protein, suchas provided in the following GenBank Number NP_001032720. Two ligandsfor CTLA-4 have been identified, B7.1 (also known as CD80) and B7.2(also known as CD86). According to a specific embodiment the B7.1protein refers to the human protein, such as provided in the followingGenBank Number NP_005182. According to a specific embodiment the B7.2protein refers to the human protein, such as provided in the followingGenBank Number NP_001193853.

CTLA4 is expressed on both activated CD4+helper and CD8+cytotoxiceffector T cells, however the major physiological role of CTLA4 seems tobe through distinct effects on the two major subsets of CD4+ T cells:down-modulation of helper T cell activity and enhancement of regulatoryT cell immunosuppressive activity.

Typically, Naive and memory T cells express high levels of cell surfaceCD28 but do not express CTLA4 on their surface. After the TCR istriggered by antigen encounter, CTLA4 is transported to the cellsurface. Binding of any of the CTL4 ligands to CTLA4 on effector cellresults in the inhibition of IL-2 synthesis and progression through thecell cycle and termination of T-cell responses [Walunas et al., J. Exp.Med. 183: 2541-2550 (1996); Greenwald et al., Immunity 14: 145-155(2001)]. Regulatory T cells, on the contrary, express CTLA4constitutively and CTLA4 engagement on regulatory T cell enhances itssuppressive function [Pardon (2012) Nature Reviews Cancer 12, 252-264].

Thus, CTLA4 blockade may enhance the activity of effector CD4+ T cellactivity and/or inhibit regulatory T cell-dependent immunosuppression.

As used herein, the term “CTLA4 antagonist” refers to an antagonisticagent that prevents and/or inhibits the biological function and/orexpression of CTLA4.

According to specific embodiments, the CTLA4 antagonist prevents and/orinhibits signaling to an immune cell (e.g. T cells) by CTLA4; therebysuppresses CTLA4 immune-suppressive activity.

According to specific embodiments, the CTLA4 antagonist promotes immuneresponse of a helper T cell following TCR activating signal.

According to specific embodiments, the CTLA4 antagonist inhibits immunesuppressive effect of a regulatory T cell.

According to specific embodiments, the CTLA4 antagonist of the presentinvention binds directly CTLA4 and/or binds ligands of CTLA4 andinterferes with and/or inhibits the binding of the ligands to CTLA4.

According to other specific embodiments, the CTLA4 antagonist indirectlybinds CTLA4 by acting through an intermediary molecule, for example theantagonist binds to or modulates a molecule that in turn binds to ormodulates CTLA4.

According to specific embodiments, the CTLA4 antagonist binds CTLA4.

According to other specific embodiments, the CTLA4 antagonist binds atleast one of the CTLA4 ligands (e.g. B7.1 and B7.2).

In certain embodiments, the CTLA4 antagonist exhibits one or moredesirable functional properties, such as high affinity binding to CTLA4or its ligand, e.g., binding to human CTLA4 with a K_(D) of 10⁻⁷ M, 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M or less; lack of significant cross-reactivity toother immune-check point proteins, e.g., CD28 and ICOS; the ability tostimulate T cell proliferation; the ability to increase IFN-y and/orIL-2 secretion; the ability to inhibit binding of one or more CTLA4ligands (e.g., B7.1 and B7.2) to CTLA4; the ability to inhibitregulatory T cell response; and/or the ability to inhibit growth oftumor cells.

According to a specific embodiment, the CTLA4 antagonist is an antibody.

According to specific embodiments, the CTLA4 antagonist is an anti-CTLA4antibody. Anti-CTLA4 antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognizedanti-CTLA4 antibodies can be used.

Examples of anti-CTLA4 antibodies are disclosed for example in Hurwitzet al. (1998) Proc. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho etal. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibodyCP-675206); and Mokyr et al. (1998) Cancer Res. 58:5301-5304; U.S. Pat.Nos. 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,207,156; 6,682,736;6,984,720; 5,977,318; 7,109,003; 7,132,281; 8,993,524 and 7,605,238,U.S. Patent Application Publication Nos. 09/644,668; 2005/0201994;2002/086014, International Application Publication Nos. WO2014066834;WO01/14424 and WO00/37504; WO2002/0039581; WO98/42752; WO00/37504;WO2004/035607; and WO01/14424, and European Patent No. EP1212422B1,which are hereby incorporated by reference in their entirety.

Specific anti-CTLA4 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

Ipilimumab (also known as 10D1, MDX-D010), marketed by BMS as Yervoy™, afully human monoclonal IgG antibody that binds to CTLA-4; and

Tremelimumab, (ticilimumab, CP-675,206, produced by Medlmmune andPfizer), a human IgG2 monoclonal antibody that binds CTLA4.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to CTLA4.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on CTLA4 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

Other CTLA4 antagonists that can be used according to some embodimentsof the invention include nucleotides, expression vectors, smallmolecules, peptides, fusion proteins and fragments targeting CTL4 or anyof its ligands, non-functional CTLA4, soluble CTLA4 or fragments thereofthat bind to CTLA4 ligands and prevent binding to the endogenous CTLA4receptor, such as disclosed for examples in U.S. Pat. No. 8,993,524,U.S. Patent Application Publication Nos. 20030232323; 20020106730; and20140271677, International Patent Application Publication Nos.WO2014012479; and WO2014089113, which are hereby incorporated byreference in their entirety. According to an aspect of the presentinvention there is provided a method of treating cancer in a subject inneed thereof, the method comprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aLAG-3 antagonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aLAG-3 antagonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a LAG-3 antagonist; and a pharmaceuticallyacceptable carrier or diluent.

LAG-3 (also known as CD223) is a member of the immunoglobulinsuperfamily which is expressed on the surface of several immune cellssuch as activated T cells, natural killer cells, B cells andplasmacytoid dendritic cells.

According to a specific embodiment the LAG-3 protein refers to the humanprotein, such as provided in the following GenBank Number NP_002277. Theonly currently known ligand for LAG-3 is MHC class II molecules, whichare upregulated on tumor-infiltrating macrophages and dendritic cellsand also on some cancers such as epithelial cancers [Pardon (2012)Nature Reviews Cancer 12, 252-264].

Upon ligand binding LAG-3 inhibits e.g. effector T cell activation,proliferation and function and also has a role in enhancing the functionof regulatory T cells. The LAG-3 MHC class II interaction has also rolesin modulating dendritic cell function. Thus, LAG3 blockade may enhancethe activity of effector T cell activity and inhibit regulatory T cellcell-dependent immunosuppression.

As used herein, the term “LAG-3 antagonist” refers to an antagonisticagent that prevents and/or inhibits the biological function and/orexpression of LAG-3.

According to specific embodiments, the LAG-3 antagonist prevents and/orinhibits signaling to an immune cell (e.g. T cells) by LAG-3; therebysuppresses LAG-3 immune-suppressive activity.

According to specific embodiments, the LAG-3 antagonist promotes immuneresponse of an effector T cell following TCR activating signal.

According to specific embodiments, the LAG-3 antagonist of the presentinvention binds directly LAG-3 and/or binds ligands of LAG-3 andinterferes with and/or inhibits the binding of the ligands to LAG-3.

According to other specific embodiments, the LAG-3 antagonist indirectlybinds LAG-3 by acting through an intermediary molecule, for example theantagonist binds to or modulates a molecule that in turn binds to ormodulates LAG-3.

According to specific embodiments, the LAG-3 antagonist binds LAG-3.

In certain embodiments, the LAG-3 antagonist exhibits one or moredesirable functional properties, such as high affinity binding to LAG-3,e.g., binding to human LAG-3 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M,10⁻¹⁰ M or less; lack of significant cross-reactivity to otherimmune-check point proteins; the ability to stimulate T cellproliferation; the ability to increase IFN-y, IL-4 and/or IL-2secretion; the ability to inhibit binding of MHC class II to LAG-3;and/or the ability to inhibit growth of tumor cells.

According to a specific embodiment, the LAG-3 antagonist is an antibody.

According to specific embodiments, the LAG-3 antagonist is an anti-LAG-3antibody. Anti-LAG-3 antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognizedanti-LAG-3 antibodies can be used. Examples of anti-LAG-3 antibodies aredisclosed for example in Blackburn et al. (2009) Nat Immunol. 10(1):29-37, Woo et al. (2012) Cancer Res, 72: 917-927, U.S. Patent Nos.6,143,273; 5,955,300; and RE38313, U.S. Application Publication No.20110150892, International Application Publication Nos. WO2014008218;WO2014144666; WO2003035682; WO2011109789; and WO2014140180, which arehereby incorporated by reference in their entirety.

Specific anti-LAG-3 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

BMS-986016 (produced by BMS), a monoclonal antibody that binds LAG-3;and

IMP701 (produced by Immutep), an antagonist antibody that binds LAG-3.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to LAG-3.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on LAG-3 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

Other LAG-3 antagonists that can be used according to some embodimentsof the present invention include nucleotides, expression vectors, smallmolecules, peptides, fusion proteins and fragments targeting LAG-3,non-functional LAG-3 (such as LAG-3 molecule lacking the intracellularKIEELE domain), soluble LAG-3 or fragments thereof that bind to a LAG-3ligand and prevent binding to the endogenous LAG-3 receptor, such asdisclosed for example in Goldberg and Drake (2011) Curr Top MicrobiolImmunol. 344:269-78, EP Patent No. EP 0893507, U.S. Pat. Nos.: 6,482,925and RE38313; U.S. Application Publication No. 20140271677, InternationalApplication Publication Nos: WO2003035682; WO2014012479; andWO2011109789, which are hereby incorporated by reference in theirentirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aTIM-3 antagonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aTIM-3 antagonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a TIM-3 antagonist; and a pharmaceuticallyacceptable carrier or diluent.

TIM-3 is a type I transmembrane protein having a structurally conservedimmunoglobulin variable (IgV) domain and a mucin domain expressed on thesurface of several immune cells such as T cells (mainly Thl cells)macrophages and dendritic cells. According to a specific embodiment theTIM-3 protein refers to the human protein, such as provided in thefollowing GenBank Number NP_116171. A ligand for TIM-3 have beenidentified, glectin-9. It has been shown that galectin 9 is upregulatedin various types of cancer, including breast cancer [Pardon (2012)Nature Reviews Cancer 12, 252-264]. According to a specific embodimentthe galectin-9 protein refers to the human protein, such as provided inthe following GenBank Numbers NP_002299 and NP_033665.

Binding of TIM-3 to its ligand negatively regulates Thl cell immunity byspecifically inducing cell death in effector Thl cells.

As used herein, the term “TIM-3 antagonist” refers to an antagonisticagent that prevents and/or inhibits the biological function and/orexpression of TIM-3.

According to specific embodiments, the TIM-3 antagonist prevents and/orinhibits signaling to an immune cell (e.g. T cells, e.g. Thl cells) byTIM-3; thereby suppresses TIM-3 immune-suppressive activity.

According to specific embodiments, the TIM-3 antagonist promotes immuneresponse of an effector T cell (e.g. Th1 cells) following TCR activatingsignal.

According to specific embodiments, the TIM-3 antagonist of the presentinvention binds directly TIM-3 and/or that binds ligands of TIM-3 andinterferes with and/or inhibits the binding of the ligands to TIM-3.

According to other specific embodiments, the TIM-3 antagonist indirectlybinds TIM-3 by acting through an intermediary molecule, for example theantagonist binds to or modulates a molecule that in turn binds to ormodulates TIM-3.

According to specific embodiments, the TIM-3 antagonist binds TIM-3.

According to other specific embodiments, the TIM-3 antagonist binds aTIM-3 ligand (e.g. galectin-9).

In certain embodiments, the TIM-3 antagonist exhibits one or moredesirable functional properties, such as high affinity binding to TIM-3,e.g., binding to human TIM-3 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M,10⁻¹⁰ M or less; lack of significant cross-reactivity to otherimmune-check point proteins; the ability to increase Thl mediatedimmune-response; the ability to stimulate T cell proliferation; theability to increase IFN-γ secretion; the ability to inhibit binding ofgalectin-9 to TIM-3; and/or the ability to inhibit growth of tumorcells.

According to a specific embodiment, the TIM-3 antagonist is an antibody.

According to specific embodiments, the TIM-3 antagonist is an anti-TIM-3antibody. Anti-TIM-3 antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognizedanti-TIM-3 antibodies can be used. Examples of anti-TIM3 antibodies aredisclosed for example in Ngiow et al. (2011) Cancer Res 71:3540-51;Sakuishi et al.(2010) Exp Med 207: 2187-94 U.S. Pat. Nos. 8,647,623;8,841,418; 8,709,412, U.S. Patent Application Publication No.20120189617, International Application Publication Nos. WO2011159877;WO2005033144 and WO2014022332, which are hereby incorporated byreference in their entirety.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to TIM-3.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on TIM-3 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

According to a specific embodiment, the TIM-3 antagonist is ananti-galectin-9 antibody.

Other TIM-3 antagonists that can be used according to specificembodiments of the present invention include nucleotides, expressionvectors, small molecules, peptides, fusion proteins and fragmentstargeting TIM-3 or it's ligand, such as disclosed for examples in U.S.Pat. No. 8,709,412, U.S. Application Publication Nos: 20140271677;20100061992, International Patent Application Publication Nos:WO2014022332; and WO2005033144, which are hereby incorporated byreference in their entirety.

It has also been suggested that CEACAM1 is a TIM-3 ligand, thus a TIM-3antagonist can be an agent that prevents signaling mediated by theinteraction of TIM-3 with CEACAM1 (see International Patent ApplicationPublication No. WO2014022332).

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aKIR antagonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aKIR antagonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a KIR antagonist; and a pharmaceuticallyacceptable carrier or diluent.

KIRs (killer cell Ig-like receptors) are cell surface glycoproteins,comprising one to three extracellular immunoglobulin-like domains, whichare expressed by some T cells as well as most human NK cells. KIRsinteract with determinants in the MHC class I molecules and uponinteraction KIRs deliver an inhibitory or an activating signal. KIRgenes are characterized by the number of Ig domains (2D or 3D) and bythe length of their cytoplasmic tail: Long-tailed KIRs (2DL or 3DL)contain immuno-receptor tyrosine-based inhibition motifs (ITIMs), whichrecruit the phosphatase SHP-1 upon receptor engagement and induceinhibitory signals; Short-tailed KIRs (2DS or 3DS) lack ITIMs and sendactivating signals to NK cells by association with the adaptor signalingmolecule DAP12 via a charged amino acid in the transmembrane region.

A number of KIRs are well characterized (see, e.g., Carrington andNorman, The KIR Gene Cluster, May 28, 2003, available through theNational Center for Biotechnology Information (NCBI) web site atwww(dot)ncbi(dot)nlm(dot)nih(dot)gov/books/bookres(dot)fcgi/mono_003/ch1d1(dot)pdf).

The sequences of human KIR genes and cDNAs, as well as their proteinproducts, are available in public databases, including GenBank.Non-limiting exemplary GenBank entries of human KIRs have the followingaccession numbers: KIR2DL1: Genbank accession number U24076, NM_014218,AAR16197, L41267 or NP_055033; KIR2DL2: Genbank accession number U24075,L76669 or NP_055034; KIR2DL3: Genbank accession number U24074, L41268 orNP_056952; KIR2DL4: Genbank accession number X97229; and KIR3DL1:Genbank accession number L41269.

KIRs appear to interact with different subsets of MHC antigens dependingupon the KIR subtype. In humans, for example, KIRs having two Ig domains(KIR2D) recognize HLA-C allotypes, KIR2DL2 and KIR2DL3 recognize anepitope shared by group 1 HLA-C allotypes, KIR2DL1 recognizes an epitopeshared by the reciprocal group 2 HLA-C allotypes, and KIR3DL2 recognizesHLA-A3 and -A11. Typically, an individual cell (e.g. NK cell) expressesseveral different KIRs and specifically binds to certain MHC allotypes.

As used herein, the term “KIR” refers to a KIR that delivers aninhibitory signal to the cell. Typically, the inhibitory KIR has two orthree extracellular Ig domains with a long intra-cytoplasmic tail(KIR2DL, KIR3DL).

According to specific embodiments, the KIR protein refers to KIR2DL1,KTR2DL2 and/or KTR2DL3.

As used herein, the term “KIR antagonist” refers to an antagonisticagent that prevents and/or inhibits the biological function and/orexpression of KIR.

According to specific embodiments, the KIR antagonist prevents and/orinhibits signaling to an immune cell (e.g. NK cells) by KIR; therebysuppresses KIR immune-suppressive activity.

According to specific embodiments, the KIR antagonist promotes immuneresponse of a NK cell.

The KIR antagonist of the present invention encompasses an antagonistthat binds directly KIR and/or that binds ligands of KIR (e.g. NHC classI) and interferes with and/or inhibits the binding of the ligand to KIR.

According to specific embodiments, the KIR antagonist binds KIR.

According to a specific embodiment, the KIR antagonist binds a specificKIR with no cross-reactivity to other KIRs.

According to a specific embodiment, the KIR antagonist binds at leasttwo, at least three different KIRs (e.g. human KIR receptors KIR2DL1,KIR2DL2, KIR2DL3).

According to other specific embodiments, the KIR antagonist binds atleast one of an MHC class I allotype.

According to other specific embodiments, the MR antagonist indirectlybinds KIR by acting through an intermediary molecule, for example theantagonist binds to or modulates a molecule that in turn binds to ormodulates KIR.

In certain embodiments, the KIR antagonist exhibits one or moredesirable functional properties, such as high affinity binding to KIR,e.g., binding to human KIR2DL1, KTR2DL2 and/or KTR2DL3 with a K_(D) of10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M or less; lack of significantcross-reactivity to other immune-check point proteins; the ability tostimulate NK cell cytotoxicity and/or the ability to inhibit growth oftumor cells.

According to a specific embodiment, the KIR antagonist is an antibody.

According to specific embodiments, the KIR antagonist is an anti-KIRantibody.

According to a specific embodiment, the anti-KIR antibody specificallybinds one of the KIR receptors.

Anti-KIR antibodies suitable for use in the invention can be generatedusing methods well known in the art especially in light of the detaileddescription hereinabove. Alternatively, art recognized anti-KIRantibodies can be used. Examples of anti-KIR antibodies are disclosedfor example in Romagne et al. (2009) Blood. 114: 2667-77, Vey et al.(2009) Blood 114: 632, International Patent Application Publication Nos.WO2014012479; WO2012160448; WO2006072625; WO2006003179; WO2006072626;WO2005003172; WO2005003168; WO2004056392; WO2005105849; WO2005009465;WO2005079766; WO2005003168; WO2005003172; WO2005037306 and WO2012160448,U.S. Patent Application Publication Nos. 20100189723; 20050037002, andU.S. Patent No. 8,637,258, which are hereby incorporated by reference intheir entirety.

Specific anti-KIR antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

Lirilumab (also known as IPH2102; BMS-986015, produced by BMY), a fullyhuman monoclonal antibody that binds KIR, specifically KIR2DL1/2/3; and

IPH2101 (also known as 1-7F9, produced by Innate Pharma), a fully humanIgG4 anti- KIR monoclonal antibody that binds KIR, specifically,KIR2DL1/2/3.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to KIR.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on KIR as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

Other KIR antagonists that can be used according to specific embodimentsof the invention include nucleotides, expression vectors, smallmolecules, peptides, fusion proteins and fragments targeting KIR, suchas disclosed for examples in U.S. Application Publication No.20140271677; International Patent Application Publication No.WO2014012479; WO2012160448; and WO2006050270, and U.S. PatentApplication Publication No. 20110091482, which are hereby incorporatedby reference in their entirety.

Specific antagonistic peptides that can be used according to someembodiments of the present invention are VAPWNDAL and VAPWSNDYL, whichwere shown to be inhibitors of KIR2DL3 [Fadda et al. (2010) Proc NatlAcad Sci U S A, 107(22):10160-5; and Gwenoline et al. (2013) J Immunol.190(6): 2924-2930].

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to said subject a therapeutically effective amount ofa IDO antagonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aIDO antagonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a IDO antagonist; and a pharmaceuticallyacceptable carrier or diluent.

IDO (indoleamine 2,3-dioxygenase), EC 1.13.11.52, is a heme-containingintracellular enzyme that catalyzes the first and rate-determining stepin the degradation of the essential amino acid L-tryptophan toN-formyl-kynurenine.

According to a specific embodiment, the IDO protein refers to the humanprotein, such as provided in the following GenBank Number NP_002155.

Tryptophan is an amino acid which is essential for cell proliferationand survival. By locally depleting tryptophan and increasingproapoptotic kynurenines, IDO expressed by e.g. dendritic cells or tumorcells can affect immune cell (e.g. T-cell) proliferation and survival.

As used herein, the term “IDO antagonist” refers to an antagonisticagent that prevents and/or inhibits the biological function and/orexpression of IDO. The term also encompasses antagonists of the IDOisoenzymes, including for example TDO (tryptophan (2,3)-dioxygenase)and/or ID02. Thus, the IDO antagonist for use in the present inventionmay inhibit, directly or indirectly, IDO and/or TDO and/or ID02.

Methods of evaluating IDO enzymatic activity are well known in the artand include e.g. measurement of the production kynurenine (thehydrolysis product of N-formyl-kynurenine) from tryptophan (see e.g.Daubener, W., et al. (1994) J. Immunol. Methods 168:39-47).

According to specific embodiments, the IDO antagonist prevents and/orinhibits deprivation of tryptophan and/or elevation of kyurenine from animmune cell (e.g. T cells); thereby prevents and/or inhibits thesuppressive activity of IDO on an immune cell (e.g. T cells).

According to specific embodiments, the IDO antagonist binds IDO.

According to other specific embodiments, the IDO antagonist indirectlybinds IDO by acting through an intermediary molecule, for example theantagonist binds to or modulates a molecule that in turn binds to ormodulates IDO.

The IDO antagonist may be a reversible or an irreversible antagonist.

According to a specific embodiment, the IDO antagonist is a reversibleantagonist i.e. reversibly inhibits IDO enzyme activity either at thecatalytic site or at a non-catalytic site.

According to another specific embodiment, the IDO antagonist is anirreversible antagonist i.e. irreversibly destroys IDO enzymaticactivity by forming a covalent bond with the enzyme.

Suitable IDO inhibitors include those based on natural products, such asthe cabbage extract bras sinin, the marine hydroid extract annulin B andthe marine sponge extract exiguamine A, including synthetic derivativesthereof.

According to specific embodiments, the IDO antagonist is a smallmolecule. Such inhibitors include the tryptophan mimetic 1-methyltryptophan (1-MT, see e.g. U.S. Pat. No. 8,383,613, the content of whichare incorporated herein in its entirety). 1-MT occurs as twostereoisomers: the L isomer significantly inhibits IDOL, while the Disomer is more specific for ID02. Examples of other small moleculestargeting IDO include, but are not limited to, oxadiazole and otherheterocyclic IDO inhibitors which are disclosed in U.S. PatentApplication Publication Nos. 20060258719 and 20070185165,alpha-methyl-tryptophan (disclosed in e.g. International PatentApplication Publication No. WO2011100295), 1-methyl-DL-tryptophan,p-(3-benzofuranyl)-DL-alanine, p-[3-benzo(b)thienyl]-DL-alanine, and6-nitro-L-tryptophan) [disclosed e.g. in Munn et al. (1999)],hydroxyanthranilic acid (disclosed e.g. in International PatentApplication Publication No. WO2009063241), INCB24360 (a hydroxyamidinesmall-molecule inhibitor) and cannabinoids (disclosed e.g. in EuropeanPatent No. EP2341903). Other small molecules that can be used as IDOinhibitors are disclosed in International Patent Application PublicationNos. WO2014150677; WO2014150646; WO2014066834; WO2012142237;WO2008115804 and WO2004094409; WO2014081689; WO2008068621; WO2000066764,U.S. Patent Application Publication Nos. 20110053941, and U.S. Pat. Nos.8,088,803; 8,748,469; 8,389,568; 7,799,776; 8,476,454; and 7,705,022,which are incorporated herein in their entirety.

Specific small molecules that can be used according to some embodimentsof the present invention include, but are not limited to:

NLG919 (RG6078, produced by Roche);

F001287 (produced by BMY);

Indoximod (D-1MT/ NLG8189, produced by NewLink Genetics);

NLG-919 (produced by NewLink Genetics); and

INCB-024360 (produced by Incyte).

Other IDO antagonists that can be used according to specific embodimentsof the present invention include, but are not limited to, nucleotides,expression vectors, small molecules, peptides, antibodies, fusionproteins and fragments targeting IDO.

Exemplary expression vectors that can be used are disclosed in U.S.Application Publication No. 20140271677, which is hereby incorporated byreference it's their entirety.

Exemplary siRNA that can be used is disclosed in U.S. Pat. No.8,389,708, which is hereby incorporated by reference it's theirentirety.

Exemplary peptides that can be used are disclosed in U.S. Pat. No.8,658,603, which is hereby incorporated by reference it's theirentirety.

As the immune-check point protein can be a co-stimulatory protein,according to specific embodiments, wherein the immune-check protein is aco-stimulatory protein, the CXCR4 antagonistic peptides of the presentinvention are administered in combination with an immune-check pointagonist. Following is a list of combinations that may be used inaccordance with the present teachings.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aOX40 agonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aOX40 agonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a OX40 agonist; and a pharmaceuticallyacceptable carrier or diluent.

OX40 (also known as CD134, TNFRSF4) is a co-stimulatory receptor whichbelongs to the TNF receptor super family, expressed on the surface ofseveral immune cells such as activated CD4+ and CD8+ TCR T cells.According to a specific embodiment, the OX40 protein refers to the humanprotein, such as provided in the following GenBank Number NP_003318. Aligand for OX40 has been identified, OX4OL (also known as CD134L, CD252,TNFSF4). According to a specific embodiment, the OX4OL protein refers tothe human protein, such as provided in the following GenBank NumberNP_003317.

As used herein, the term “OX40 agonist” refers to an agonistic agentthat induces and/or increases the biological function and/or expressionof OX40.

According to specific embodiments, the OX40 agonist induces and/orincreases signaling to an immune cell (e e.g. T cells) by OX40; therebyinduces and/or increases OX40 immune co-stimulatory activity.

According to specific embodiments, the OX40 agonist promotes immuneresponse of an effector T cell (e.g. CD4+ and CD8+ T cell) following TCRactivating signal.

According to specific embodiments, the OX40 agonist binds and activatesOX40.

According to specific embodiments, the OX40 agonist binds ligands ofOX40 and increases the binding (e.g. affinity) of the ligands to OX40and/or activation of OX40 by the ligand.

According to other specific embodiments, the OX40 agonist indirectlybinds OX40 by acting through an intermediary molecule, for example theagonist binds to or modulates a molecule that in turn binds to ormodulates OX40.

In certain embodiments, the OX40 agonist exhibits one or more desirablefunctional properties, such as high affinity binding to OX40, e.g.,binding to human OX40 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M orless; lack of significant cross-reactivity to other immune-check pointproteins; the ability to stimulate T cell differentiation and/orproliferation; the ability to increase IFN-γ and/or IL-2 secretion; theability to increase T cell cytotoxicity; the ability to generate memoryT cells; the ability to inhibit regulatory T cell function and/or theability to inhibit growth of tumor cells.

According to a specific embodiment, the OX40 agonist is the naturallyoccurring ligand (e.g. OX4OL) or a functional derivative or variantthereof which retain the ability to specifically bind to the OX40. Thus,example, the OX40 agonist can be a entire OX4OL, soluble OX4OL orfragments thereof (e.g. a soluble molecule comprising the extracellularOX4OL domains) and fusion proteins comprising a functionally activeportion of OX4OL covalently linked to a second protein domain, thatbinds to and activates OX40, such as described in U.S. Pat. Nos.5,457,035; 7,622,444; 6,312,700 and International Patent ApplicationPublication No. WO95/21915; which are hereby incorporated by referencein their entirety.

According to a specific embodiment, the OX40 agonist is an antibody.

According to specific embodiments, the OX40 agonist is an anti-OX40antibody. Anti-OX40 antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognizedanti-OX40 antibodies can be used. Examples of anti-OX40 antibodies aredisclosed for example in Weinberg, A. et al. (2000) Immunol 164:2160-2169, Weinberg, A. D., et al. (2006) J Immunother 29, 575-585,International Patent Application Publication Nos. WO2011109789;WO2013038191; WO95/12673 and WO95/21915, and U.S. Patent No. 7,504,101,which are hereby incorporated by reference in their entirety.

Specific anti-OX40 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

MOXR0916 (also known as RG7888, produced by Roche), a humanizedmonoclonal antibody that binds OX40;

MEDI0562 (produced by AZY/Medlmmune), a humanized monoclonal antibodythat binds OX40;

MEDI6469 (produced by AZY/Medlmmune), a murine-based antibody that bindsOX40; and

9B12 (described in Weinberg, A. D., et al. (2006) J. Immunother 29,575-585], a murine IgG1 monoclonal antibody directed against theextracellular domain of human OX40.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to OX40.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on OX40 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

According to other specific embodiments, the OX40 agonist is a fusionprotein in which one or more domains of OX4OL is covalently linked toone or more additional protein domains. Exemplary OX4OL fusion proteinsthat can be used as OX40 agonists are described in U.S. Pat. Nos.6,312,700; 7,622,444, International Patent Application Publication Nos.WO2011109789; and WO2010105068, the disclosures of which areincorporated herein by reference in their entirety.

According to specific embodiments, the OX40 agonist includes an OX4OLfusion polypeptide that self- assembles into a multimeric (e.g.,trimeric or hexameric) OX4OL fusion protein. Such fusion proteins aredescribed, e.g., in Morris et al. (2007) Mol Immunol. 44(12): 3112-3121,U.S. Patent No. 7,959,925, which is incorporated by reference herein inits entirety.

According to specific embodiments, the OX40 agonist is a OX40polypeptide agonist linked to an agonistic polypeptide of anotherco-stimulatory check point protein, for example, a polypeptide agonistfor OX40 linked to a polypeptide agonist for CD40 or CD137, such asdisclosed in International Patent Application Publication No.WO2014121099 and WO2012109203, respectively, which are incorporated byreference herein in their entirety.

A specific fusion protein that can be used according to some embodimentsof the present invention is MEDI6383 (produced by AZY/Medlmmune), ahuman OX40 ligand fusion protein.

Other OX40 agonists that can be used according to specific embodimentsof the present invention include nucleotides, expression vectors andpeptides, such as disclosed for example in Linch et al. (2015) FrontOncol. 5: 34, U.S. Pat. No. 6,312,700 and U.S. Application PublicationNo. 20140271677, which are hereby incorporated by reference in theirentirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCD137 agonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aCD137 agonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD137 agonist; and a pharmaceuticallyacceptable carrier or diluent.

CD137 (also known as 4-1BB and TNFRSF9), a member of the tumor necrosisfactor receptor superfamily of co-stimulatory molecules, is a membraneglycoprotein that is inducibly expressed on several immune cells such asactivated T cells, B cells, dendritic cells and natural killer (NK)cells. According to a specific embodiment the CD137 protein refers tothe human protein, such as provided in the following GenBank NumberNP_001552. A ligand for CD137 have been identified, CD137L (also knownas 4-1BBL and TNFSF9), which is expressed on activatedantigen-presenting cells, myeloid progenitor cells, and hematopoieticstem cells.

According to a specific embodiment the CD137L protein refers to thehuman protein, such as provided in the following GenBank NumberNP_003802.

The interaction of CD137 with its ligand has been shown to co-stimulateproliferation of T lymphocytes, enhances B cells proliferation andimmunoglobulin synthesis, also induces proliferation of monocytes.

As used herein, the term “CD137 antagonist” refers to an agonistic agentthat induces and/or increases the biological function and/or expressionof CD137.

According to specific embodiments, the CD137 agonist induces and/orincreases signaling to an immune cell (e.g. T cells) by CD137; therebyinduces and/or increases CD137 immune co-stimulatory activity.

According to specific embodiments, the CD137 agonist promotes immuneresponse of an effector T cell following TCR activating signal.

According to specific embodiments, the CD137 agonist binds directlyCD137 and activates the receptor.

According to specific embodiments, the CD137 agonist binds ligands ofCD137 and increases the binding (e.g. affinity) of the ligands to CD137and/or activation of CD137 by the ligand.

According to other specific embodiments, the CD137 agonist indirectlybinds CD137 by acting through an intermediary molecule, for example theagonist binds to or modulates a molecule that in turn binds to ormodulates CD137.

In certain embodiments, the CD137 agonist exhibits one or more desirablefunctional properties, such as high affinity binding to CD137, e.g.,binding to human CD137 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ Mor less; lack of significant cross-reactivity to other immune-checkpoint proteins; the ability to stimulate T cell proliferation; theability to increase IFN-y secretion; the ability to increase T cellscytotoxic activity; the ability to prevent activation induced celldeath; and/or the ability to inhibit growth of tumor cells.

According to a specific embodiment, the CD137 agonist is the naturallyoccurring ligand (e.g. CD137L) or a functional derivative or variantthereof which retain the ability to specifically bind to the CD137.Thus, for example the GITR agonist can be an entire CD137L, solubleCD137L or fragments thereof and fusion proteins comprising afunctionally active portion of CD137L covalently linked to a secondprotein domain, that binds to and activates CD137.

According to a specific embodiment, the CD137 agonist is an antibody.

According to specific embodiments, the CD137 agonist is an anti-CD137antibody. Anti-CD137 antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognizedanti-CD137 antibodies can be used. Examples of anti-CD137 antibodies aredisclosed for example in Melero, I. et al. (1997) Nature Medicine 3:682-685 (1997), International Patent Application Publication Nos.WO2014144666; WO2006088447; WO2006088464; U.S. Patent ApplicationPublication Nos. 20080166336; 20050095244, U.S. Pat. Nos. 7,214,493;6,887,673; 8,337,850; 8,821,867; 7,288,638; 6,303,121; 6,569,997;6,905,685; 6,355,476; 6,362,325; 6,974,863; 6,210,669; 5,928,893, whichare hereby incorporated by reference in their entirety.

Specific anti-CD137 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

BMS-666513 (also known as urelumab, produced by BMY), a fully human IgG4monoclonal antibody that binds human CD137;

BMS-663031, BMS-469492; or BMS-469497; (produced by BMY, described inU.S. Pat. Nos. 7,288,638; and 6,362,325;

XmAb-5592 (Xencor); a Fc-engineered and humanized anti-CD137 antibody;and

PF-05082566 (produced by Pfizer), a fully human IgG2 monoclonal antibodythat binds to the extracellular domain of human CD137.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to CD137.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on CD137 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

According to other specific embodiments, the CD137 agonist is a fusionprotein in which one or more domains of CD137L is covalently linked toone or more additional protein domains. Exemplary CD137 fusion proteinsthat can be used as CD137 agonists are described in InternationalApplication Publication No. WO2014012479; WO2011109789; WO2012109203,which are hereby incorporated by reference in their entirety.

According to specific embodiments, the CD137 agonist is a CD137polypeptide agonist linked to an agonistic polypeptide of anotherco-stimulatory check point protein, for example, a polypeptide agonistfor CD137 linked to a polypeptide agonist for e.g. OX40, CD40, ICOS,CD28, CD27, CD70 and GITR such as disclosed in International PatentApplication Publication No. WO2012109203, which is incorporated byreference herein in its entirety.

According to other specific embodiments, the CD137 agonist is anaptamer. Exemplary aptamers that can be used as CD137 agonists aredescribed in McNamara et al. (2008) J. Clin. Invest. 1 18: 376-386 andInternational Application Publication No. WO2007035518, which are herebyincorporated by reference in their entirety.

Other CD137 agonists that can be used according to some embodiments ofthe present invention include nucleotides and expression vectors such asdisclosed for examples in U.S. Application Publication No. 20140271677,which is hereby incorporated by reference in its entirety.

It has also been suggested that galectin-9 is a CD137 ligand, thus aCD137 agonist can be an agent that promotes signaling mediated by theinteraction of CD137 with galectin-9 (see International PatentApplication Publication No. WO2012177788).

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCD27 agonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aCD27 agonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD27 agonist; and a pharmaceuticallyacceptable carrier or diluent.

CD27 (also known as TNFRSF7, S152), a member of the tumor necrosisfactor receptor superfamily, is a type I transmembrane protein expressedon the surface of several immune cells such as T cells, B cells and NKcells. According to a specific embodiment, the CD27 protein refers tothe human protein, such as provided in the following GenBank NumberNP_001233. A ligand for CD27 has been identified, CD70 (also known asTNFSF7). CD70 is transiently expressed following cell activation ondendritic cells, T cells, B cells and NK cells; and also on a variety oftransformed cells including lymphoma B cells, Hodgkin's andReed-Sternberg cells, malignant cells of neural origin, and a number ofcarcinomas. According to a specific embodiment the CD70 protein refersto the human protein, such as provided in the following GenBank NumberNP_001243.

The interaction of CD27 with its ligand has been shown to activate theNF-kp signaling pathways that in turn stimulates B cell and T cellproliferation, cytokine secretion, plasma cell differentiation andsubsequent antibody secretion (see e.g. Yamamoto, H. 1998 J Immunol.161(9): 4753-9).

As used herein, the term “CD27 antagonist” refers to an agonistic agentthat induces and/or increases the biological function and/or expressionof CD27.

According to specific embodiments, the CD27 agonist induces and/orincreases signaling to an immune cell (e.g. T cells, B cells, NK cells)by CD27; thereby induces and/or increases CD27 immune co-stimulatoryactivity.

According to specific embodiments, the CD27 agonist promotes immuneresponse of an effector T cell following TCR activating signal.

According to specific embodiments, the CD27 agonist promotes immuneresponse of a B cell.

According to specific embodiments, the CD27 agonist binds directly CD27and activates the receptor.

According to specific embodiments, the CD27 agonist binds ligands ofCD27 and increases the binding (e.g. affinity) of the ligands to CD27and/or activation of CD27 by the ligand.

According to other specific embodiments, the CD27 agonist indirectlybinds CD27 by acting through an intermediary molecule, for example theagonist binds to or modulates a molecule that in turn binds to ormodulates CD27.

In certain embodiments, the CD27 agonist exhibits one or more desirablefunctional properties, such as high affinity binding to CD27, e.g.,binding to human CD27 with a KD of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M orless; lack of significant cross-reactivity to other immune-check pointproteins; the ability to stimulate T cell proliferation; the ability toincrease IFN-y, IL-4 and/or IL-2 secretion; the ability to stimulateantigen-specific memory responses; the ability to stimulate antibodyresponses and/or the ability to inhibit growth of tumor cells.

According to a specific embodiment, the CD27 agonist is the naturallyoccurring ligand (e.g. CD70) or a functional derivative or variantthereof which retain the ability to specifically bind to the CD27. Thus,for example the CD27 agonist can be an entire CD70, soluble CD70 orfragments thereof and fusion proteins comprising a functionally activeportion of CD70 covalently linked to a second protein domain, that bindsto and activates CD27.

According to a specific embodiment, the CD27 agonist is an antibody.

According to specific embodiments, the CD27 agonist is an anti-CD27antibody. Anti-CD27 antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognizedanti-CD27 antibodies can be used.

Examples of anti-CD27 antibodies are disclosed for example in Van Lieret al., 1987, J Immunol 139:1589-96, International Patent ApplicationPublication Nos. WO2012004367; and WO2008/051424, U.S. PatentApplication Publication Nos. 20120213771; and 20120093805, which arehereby incorporated by reference in their entirety.

Specific anti-CD27 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

Varlilumab (also known as CDX-1127, produced by Celldex), a fully humanmonoclonal antibody that binds CD27.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to CD27.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on CD27 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

Other CD27 agonists that can be used according to some embodiments ofthe present invention include nucleotides and expression vectors, suchas disclosed for examples in U.S. Application Publication No.20140271677, which are hereby incorporated by reference in theirentirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO:1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aCD40 agonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aCD40 agonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD40 agonist; and a pharmaceuticallyacceptable carrier or diluent.

CD40 is a co-stimulatory receptor expressed on the surface of severalimmune cells such as antigen presenting cells. According to a specificembodiment the CD40 protein refers to the human protein, such asprovided in the following GenBank Number NP_001241. A ligand for CD40has been identified, CD4OL (also known as CD154), which is mainlyexpressed on T cells. According to a specific embodiment the CD4OLprotein refers to the human protein, such as provided in the followingGenBank Number NP_000065.

The interaction of CD40 with its ligand has been shown to enhanceantigen presenting cells maturation, antigen-presenting function,co-stimulatory potential and stimulate cytolytic activity of immunecells. Specifically, CD40 engagement increases release ofimmunoregulatory cytokines such as IL-6 IL-12 IL-15, increasedexpression of MHC class I and II, and increased expression of adhesionmolecules (e.g., ICAM) and costimulatory molecules (e.g., B7).

In addition to enhancement of cellular and immune function, the effectsof CD40 activation induce apoptosis of CD40 positive cells.

As used herein, the term “CD40 agonist” refers to an agonistic agentthat induces and/or increases the biological function and/or expressionof CD40.

According to specific embodiments, the CD40 agonist induces and/orincreases signaling to an immune cell (e.g. B cell, dendritic cells) byCD40; thereby induces and/or increases CD40 immune co-stimulatoryactivity.

According to specific embodiments, the CD40 agonist promotes immuneresponse of an immune cell e.g. dendritic cell, B cell, NK cell and/oreffector T cell.

According to specific embodiments, the CD40 agonist promotes cell deathof a CD40 positive cell.

According to specific embodiments, the CD40 agonist binds directly CD40and activates the receptor.

According to specific embodiments, the CD40 agonist binds a ligand ofCD40 and increases the binding (e.g. affinity) of the ligand to CD40and/or activation of CD40 by the ligand.

According to other specific embodiments, the CD40 agonist indirectlybinds CD40 by acting through an intermediary molecule, for example theagonist binds to or modulates a molecule that in turn binds to ormodulates CD40.

In certain embodiments, the CD40 agonist exhibits one or more desirablefunctional properties, such as high affinity binding to CD40, e.g.,binding to human CD40 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M orless; lack of significant cross-reactivity to other immune-check pointproteins; the ability to increase IL-8, IL-12, IL-15, IL-18 and IL-23secretion; the ability to enhance tumor antigen processing andpresentation by dendritic cells, the ability to increase the cytolyticactivity of T cells and NK cells; the ability to induces cell death ofCD40 positive cells; the ability to stimulate antibody responses; and/orthe ability to inhibit growth of tumor cells.

According to a specific embodiment, the CD40 agonist is the naturallyoccurring ligand (e.g. CD4OL) or a functional derivative or variantthereof which retain the ability to specifically bind to the CD40. Thus,for example the CD40 agonist can be an entire CD4OL, soluble CD4OL orfragments thereof and fusion proteins comprising a functionally activeportion of CD4OL covalently linked to a second protein domain, thatbinds to and activates CD40, such as described in U.S. Pat. Nos.6,410,711; 6,391,637; 5,981,724; 5,961,974, U.S. Patent ApplicationPublication No. 20040006006, and International Application PublicationNos. WO2001016180; WO1996026735; WO1993008207, which are herebyincorporated by reference in their entirety.

According to a specific embodiment, the CD40 agonist is an antibody.

According to specific embodiments, the CD40 agonist is an anti-CD40antibody. Agonistic anti-CD40 antibodies can exert their function by atleast one of several mechanisms, including immune-enhancing effect byco-stimulation of CD40 positive cells, as well as direct killing of CD40positive cells by induction of apoptosis or by stimulating a humoralresponse leading to ADCC. Anti-CD40 antibodies suitable for use in theinvention can be generated using methods well known in the artespecially in light of the detailed description hereinabove.Alternatively, art recognized anti-CD40 antibodies can be used. Examplesof anti-CD40 antibodies are disclosed for example in Schlossman et al.,Leukocyte Typing, 1995, 1 :547-556, Hirano A. et al., Blood 93:2999-3007(1999), French R. R. et al., Nature Medicine 5:548-53 (1999), Tutt A. L.et al., J. of Immunol. 161:3176-85 (1998), Funakoshi S. et al., J. ofImmunotherapy with Emphasis on Tumor Immunol. 19:93-101 (1996),International Patent Application Publication Nos. WO2003040170;WO2005063289; WO2013034904; WO2003040170; WO2014070934; WO2012149356;WO2001037870, U.S. Patent Application Publication Nos. 20110311525;20120263732; 20120263732; 20030059427; 20090074711; 20130024956;20100098694 and U.S. Pat. Nos. 7,563,442; 7,338,660, 6,843,989,7,172,759; 7,547,438; 8,778,345; 8,388,971; 7,288,251; 7,618,633, whichare hereby incorporated by reference in their entirety.

Specific anti-CD40 antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

CP-870,893 (produced by Pfizer), a fully human IgG2 monoclonal antibodythat binds CD40;

Dacetuzumab (produced by Seattle Genetics Inc), a humanized monoclonalantibody that binds CD40;

ADC-1013 (produced by Alligator Bioscience AB), a human monoclonal IgG1anti-CD40 antibody; and

CD40.4 (5C3, produced by PharMingen).

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to CD40.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on CD40 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

According to other specific embodiments, the CD40 agonist is a fusionprotein in which one or more domains of CD4OL is covalently linked toone or more additional protein domains. According to specificembodiments, the CD40 agonist includes a multimeric CD4OL fusionpolypeptide. Exemplary CD4OL fusion proteins that can be used as CD40agonists are described in International Patent Application PublicationNos: WO2007120368, WO2001016180, the disclosures of which areincorporated herein by reference in their entirety.

According to specific embodiments, the CD40 agonist is a CD40polypeptide agonist linked to an agonistic polypeptide of anotherco-stimulatory check point protein, for example, a polypeptide agonistfor CD40 linked to a polypeptide agonist for CD40, such as disclosed inInternational Patent Application Publication No. WO2014121099, which isincorporated by reference herein in its entirety.

Other CD40 agonists that can be used according to some embodiments ofthe present invention include nucleotides, expression vectors,polypeptides, small molecules, and methods of obtaining them, such asdisclosed for examples in U.S. Application Publication Nos. 20140271677;and 20060287229 and International Application Publication No.WO2001016180, which are hereby incorporated by reference in theirentirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to the subject a therapeutically effective amount of aGITR agonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aGITR agonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a GITR agonist; and a pharmaceuticallyacceptable carrier or diluent.

GITR [glucocorticoid-induced tumor necrosis factor receptor, also knownas TNF receptor superfamily 18 (TNFRSF18)] is a type I transmembraneprotein expressed on the surface of several immune cells such as Tcells, NK cells, macrophages, B cells, dendritic cells, mast cells andmonocytes. GITR expression on T cells is upregulated upon T cellactivation. According to a specific embodiment the GITR protein refersto the human protein, such as provided in the following GenBank NumbersNP_004186, NP_683699, NP_683700. A ligand for GITR has been identified,GITRL (also known as TNFSF18). GITRL is a type II transmembrane proteinas is typical for most TNF ligand family members, expressed primarily onantigen presenting cells including macrophages, B cells, dendritic cellsand endothelial cells. According to a specific embodiment the GITRLprotein refers to the human protein, such as provided in the followingGenBank Number NP_005083.

It has been shown that the interaction of GITR with its ligand triggersa co-stimulatory signal which enhances effector T cells proliferation,survival and effector functions while inhibits the suppressive activityof regulatory T cells.

As used herein, the term “GITR agonist” refers to an agonistic agentthat induces and/or increases the biological function and/or expressionof GITR.

According to specific embodiments, the GITR agonist induces and/orincreases signaling to an immune cell (e.g. T cells) by GITR; therebyinduces and/or increases GITR immune co-stimulatory activity.

According to specific embodiments, the GITR agonist promotes immuneresponse of an effector T cell following TCR activating signal.

According to specific embodiments, the GITR agonist binds directly GITRand activates the receptor.

According to specific embodiments, the GITR agonist binds ligands ofGITR and increases the binding (e.g. affinity) of the ligands to GITRand/or activation of GITR by the ligand.

According to other specific embodiments, the GITR agonist indirectlybinds GITR by acting through an intermediary molecule, for example theagonist binds to or modulates a molecule that in turn binds to ormodulates GITR.

In certain embodiments, the GITR agonist exhibits one or more desirablefunctional properties, such as high affinity binding to GITR, e.g.,binding to human GITR with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M orless; lack of significant cross-reactivity to other immune-check pointproteins; lack of significant reactivity to other tumor necrosis factorreceptors; the ability to stimulate T cell proliferation; the ability tostimulate antibody responses and/or the ability to inhibit growth oftumor cells.

According to a specific embodiment, the GITR agonist is the naturallyoccurring ligand (e.g. GITRL) or a functional derivative or variantthereof which retain the ability to specifically bind to the GITR. Thus,for example the GITR agonist can be an entire GITRL, soluble GITRL orfragments thereof and fusion proteins comprising a functionally activeportion of GITRL covalently linked to a second protein domain, thatbinds to and activates GITR, such as described in International PatentApplication Publication No. WO2005007190, the contents of which ishereby incorporated by reference in their entirety.

According to a specific embodiment, the GITR agonist is an antibody.

According to specific embodiments, the GITR agonist is an anti-GITRantibody.

Anti-GITR antibodies suitable for use in the invention can be generatedusing methods well known in the art especially in light of the detaileddescription hereinabove. Alternatively, art recognized anti-GITRantibodies can be used. Examples of anti-GITR antibodies are disclosedfor example in International Patent Application Publication Nos.WO2005007190; WO2011109789; WO2013039954; WO2015031667, U.S. PatentApplication Publication Nos. 20070098719; 20140348841; 20140220002, U.S.Pat. Nos. 8,709,424; 8,591,886; 7,618,632; 7,812,135; and 8,388,967,which are hereby incorporated by reference in their entirety.

Specific anti-GITR antibodies that can be used according to someembodiments of the present invention include, but are not limited to:

MK-4166 (produced by Merck), a monoclonal antibody that binds GITR; and

TRX518 (produced by GITR Inc.), a humanized monoclonal antibody thatbinds human GITR.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to GITR.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on GITR as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

According to other specific embodiments, the GITR agonist is a fusionprotein in which one or more domains of GITRL is covalently linked toone or more additional protein domains. Exemplary GITRL fusion proteinsthat can be used as GITR agonists are described in International PatentApplication Publication No. WO2011109789, the contents of which areincorporated herein by reference in their entirety.

According to specific embodiments, the GITR agonist includes amultimeric GITRL fusion polypeptide, such as disclosed for examples inInternational Patent Application Publication No. WO2007120368, which isincorporated by reference herein in its entirety.

Other GITR agonists that can be used according to some embodiments ofthe present invention include nucleotides, expression vectors, smallmolecules and peptides such as disclosed for examples in U.S.Application Publication No. 20140271677, U.S. Pat. Nos. 7,618,632;8,586,023, which are hereby incorporated by reference in their entirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to said subject a therapeutically effective amount ofa CD28 agonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aCD28 agonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a CD28 agonist; and a pharmaceuticallyacceptable carrier or diluent.

CD28 is a co-stimulatory molecule expressed on the surface of severalimmune cells such as T cells. According to a specific embodiment theCD28 protein refers to the human protein, such as provided in thefollowing GenBank Numbers NP_001230006, NP_001230007, NP_006130. Severalligands for CD28 have been identified, B7.1 (also known as CD80) andB7.2 (also known as CD86). According to a specific embodiment the B7.1protein refers to the human protein, such as provided in the followingGenBank Number NP_005182. According to a specific embodiment the B7.2protein refers to the human protein, such as provided in the followingGenBank Number NP_001193853.

The interaction of CD28 with its ligand triggers a co-stimulatory signalthat synergizes with the TCR signal to promote T-cell activation,proliferation and function. CD28 signaling was shown to regulate thethreshold for T-cell activation and decrease the number of TCRengagements needed for T-cell activation.

As used herein, the term “CD28 agonist” refers to an agonistic agentthat induces and/or increases the biological function and/or expressionof CD28.

According to specific embodiments, the CD28 agonist induces and/orincreases signaling to an immune cell (e.g. T cells) by CD28; therebyinduces and/or increases CD28 immune co-stimulatory activity.

According to specific embodiments, the CD28 agonist promotes immuneresponse of an effector T cell following TCR activating signal.

According to specific embodiments, the CD28 agonist binds directly CD28and activates the receptor.

According to specific embodiments, the CD28 agonist binds ligands ofCD28 (e.g. B7.1 and B7.2) and increases the binding (e.g. affinity) ofthe ligands to CD28 and/or activation of CD28 by the ligand.

According to other specific embodiments, the CD28 agonist indirectlybinds CD28 by acting through an intermediary molecule, for example theagonist binds to or modulates a molecule that in turn binds to ormodulates CD28.

In certain embodiments, the CD28 agonist exhibits one or more desirablefunctional properties, such as high affinity binding to CD28, e.g.,binding to human CD28 with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M orless; lack of significant cross-reactivity to other immune-check pointproteins, e.g., CTLA-4 and ICOS; the ability to stimulate T cellproliferation; the ability to increase IFN-y and/or IL-2 secretion; theability to stimulate antigen-specific memory responses; the ability tostimulate antibody responses and/or the ability to inhibit growth oftumor cells.

According to a specific embodiment, the CD28 agonist is the naturallyoccurring ligand (e.g. B7.1 or B7.2) or a functional derivative orvariant thereof which retain the ability to specifically bind to theCD28. Thus, for example the CD28 agonist can be an entire B7.1 or B7.2,soluble B7.1 or B7.2 or fragments thereof and fusion proteins comprisinga functionally active portion of B7.1 or B7.2 covalently linked to asecond protein domain, that binds to and activates CD28, such asdisclosed for example in U.S. Patent Application Publication No.20030232323; and International Application Publication No. WO1995003408,which are hereby incorporated by reference in their entirety.

According to specific embodiments, the B7.1 or B7.2 functionalderivative or variant specifically binds CD28 with not cross reactivityto CTLA4.

According to a specific embodiment, the CD28 agonist is an antibody.

According to specific embodiments, the CD28 agonist is an anti-CD28antibody. The anti-CD28 antibody can be a superagonistic anti-CD28antibody or a conventional anti-CD28 antibody.

As used herein, the phrase “conventional anti-CD28 antibody” refers toan antibody which binds CD28 (e.g., in a domain outside the basolateraldomain) and co-stimulates T cells in a TCR-dependent mechanism.

As used herein, the phrase “superagonistic anti-CD28 antibody” refers toan antibody which binds CD28 through the basolateral domain resulting ina polyclonal activation of T lymphocytes even in the absence of TCRstimulation.

Anti-CD28 antibodies suitable for use in the invention can be generatedusing methods well known in the art especially in light of the detaileddescription hereinabove. Alternatively, art recognized anti-CD28antibodies can be used. Examples of anti-CD28 antibodies are disclosedfor example in Poirier et al. (2012) American Journal of Transplantation12(7): 1682-1690, Cell Immunol. 2005 Jul-Aug;236(1-2):154-60, which arehereby incorporated by reference in their entirety.

Specific anti-CD28 antibodies that can be used according to someembodiments of the present invention include, but are not limited toTAB08 (previously known as TGN1412, produced by TheraMAB), a humanizedmonoclonal antibody directed against human CD28.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to CD28.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on CD28 as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

According to other specific embodiments, the CD28 agonist is an aptamer.Exemplary aptamers that can be used as CD28 agonists are described inPastor et al. Mol Ther Nucleic Acids. (2013) Jun 11;2:e98, which ishereby incorporated by reference in its entirety.

According to specific embodiments, other CD28 agonists includenucleotides, expression vectors, small molecules, peptides, fusionproteins and fragments targeting CD28, are disclosed for examples inU.S. Application Publication Nos. 20140271677; 20040137577; 20020106730;20100303811 and International Application Publication No. WO2014089009which are hereby incorporated by reference in their entirety.

According to an aspect of the present invention there is provided amethod of treating cancer in a subject in need thereof, the methodcomprising:

(a) administering to the subject a therapeutically effective amount of apeptide having an amino acid sequence as set forth in SEQ ID NO: 1 or ananalog or derivative thereof; and

(b) administering to said subject a therapeutically effective amount ofa ICOS agonist, thereby treating the cancer in the subject.

According to an aspect of the present invention there is provided anarticle of manufacture identified for use in treating cancer, comprisinga packaging material packaging a peptide having an amino acid sequenceas set forth in SEQ ID NO: 1 or an analog or derivative thereof and aICOS agonist.

According to an aspect of the present invention there is provided apharmaceutical composition comprising as active ingredients a peptidehaving an amino acid sequence as set forth in SEQ ID NO: 1 or an analogor derivative thereof and a ICOS agonist; and a pharmaceuticallyacceptable carrier or diluent.

ICOS (also known as CD278) is a co-stimulatory molecule expressed on thesurface of several immune cells such as activated T cells. According toa specific embodiment the ICOS protein refers to the human protein, suchas provided in the following GenBank Number NP_036224. A ligand for ICOShas been identified, ICOSL (also known as B7-H2, B7RP1, CD275).According to a specific embodiment the ICOSL protein refers to the humanprotein, such as provided in the following GenBank Numbers NP_001269979,NP_001269980, NP_001269981, NP_056074. The interaction of ICOS with itsligand triggers a co-stimulatory signal that promotes T-helper celldifferentiation and effector function, and is particularly important forinterleukin-10 (IL-10) production.

As used herein, the term “ICOS agonist” refers to an agonistic agentthat induces and/or increases the biological function and/or expressionof ICOS.

According to specific embodiments, the ICOS agonist induces and/orincreases signaling to an immune cell (e.g. T cells) by ICOS; therebyinduces and/or increases ICOS immune co-stimulatory activity.

According to specific embodiments, the ICOS agonist promotes immuneresponse of an effector T cell following TCR activating signal.

According to specific embodiments, the ICOS agonist binds directly ICOSand activates the receptor.

According to specific embodiments, the ICOS agonist binds ligands ofICOS and increases the binding (e.g. affinity) of the ligands to ICOSand/or activation of ICOS by the ligand.

According to other specific embodiments, the ICOS agonist indirectlybinds ICOS by acting through an intermediary molecule, for example theagonist binds to or modulates a molecule that in turn binds to ormodulates ICOS.

In certain embodiments, the ICOS agonist exhibits one or more desirablefunctional properties, such as high affinity binding to ICOS, e.g.,binding to human ICOS with a K_(D) of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M orless; lack of significant cross-reactivity to other immune-check pointproteins, e.g., CD28 and CTLA-4; the ability to stimulate T cellproliferation; the ability to increase IL-10 secretion; and/or theability to inhibit growth of tumor cells.

According to a specific embodiment, the ICOS agonist is the naturallyoccurring ligand (e.g. ICOSL) or a functional derivative or variantthereof which retain the ability to specifically bind to the ICOS. Thus,for example the ICOS agonist can be an entire ICOSL, soluble ICOSL orfragments thereof and fusion proteins comprising a functionally activeportion of ICOSL covalently linked to a second protein domain, thatbinds to and activates ICOS, such as described in U.S. ApplicationPublication Nos. 20040137577; 20020106730, the contents of which arehereby incorporated by reference in their entirety.

According to specific embodiments, the ICOS agonist is a tumor cell ormembranes thereof expressing ICOSL that is used as an anti-tumorvaccine, such as disclosed e.g. in U.S. Patent No. 8,709,417, which ishereby incorporated by reference in its entirety.

According to a specific embodiment, the ICOS agonist is an antibody.

According to specific embodiments, the ICOS agonist is an anti-ICOSantibody. Anti-ICOS antibodies suitable for use in the invention can begenerated using methods well known in the art especially in light of thedetailed description hereinabove. Alternatively, art recognizedanti-ICOS antibodies can be used. Examples of anti-ICOS antibodies aredisclosed for example in Hutloff, A. et al. (1999) Nature 397: 262-266,Deng et al. Hybrid Hybridomics. 2004 Jun;23(3):176-82, Sakthivel et al.PLoS One. 2014 Jul 16;9(7):e100970, Redoglia et al. Eur J Immunol 199626: 2781-2789, U.S. Pat. Nos. 8,709,417; 6,803,039, InternationalApplication Publication Nos. WO2012131004; WO2014089113; WO2008137915,which are hereby incorporated by reference in their entirety.

Specific anti-ICOS antibodies that can be used according to someembodiments of the present invention include, but are not limited to anagonistic anti-ICOS antibody developed by Jounce Therapeutics.

According to a specific embodiment, the antibody competes with any ofthe above-mentioned antibodies for binding to ICOS.

According to a specific embodiment, the antibody competes for bindingwith and/or binds to the same epitope on ICOS as the above-mentionedantibodies.

According to another specific embodiment, the antibody has at leastabout 90% variable region amino acid sequence identity with theabove-mentioned antibodies.

Other ICOS agonists that can be used according to specific embodimentsof the present invention include nucleotides, expression vectors,peptides, fusion proteins and fragments targeting ICOS, functionalICOSL, are disclosed for examples in U.S. Application Publication Nos.20140271677; 20040137577; 20020106730, International ApplicationPublication No. WO2014089113, which are hereby incorporated by referencein their entirety.

The order in which the CXCR4 antagonistic peptide and the immune-checkpoint regulator are administered to the subject can vary according tothe method of treating.

Thus, according to a specific embodiment, step (a) is effected prior tostep (b).

According to another specific embodiment, step (a) is effected followingstep (b).

According to yet another specific embodiment, step (a) is effectedconcomitantly with step (b).

Multiple rounds of administration according to the methods of thepresent invention and multiple doses of the CXCR4 antagonistic peptideand the immune-check point regulator can be administered. According tospecific embodiments step (a) is effected multiple times. Thus,according to specific embodiments, administration of the immune-checkpoint regulator is effected following at least one administration of theCXCR4 antagonistic peptide. According to specific embodiments step (B)is effected multiple times. Thus, according to specific embodiments,administering the CXCR4 antagonistic peptide of the present invention iseffected following at least one administration of the immune-check pointregulator. According to specific embodiments, administering the CXCR4antagonistic peptide of the present invention is effected in asequential order with administration of the immune-check pointregulator.

According to specific embodiments, the CXCR4 antagonistic peptide can beadministered to a subject in combination with several of theimmune-check point regulators selected from the list of combinationsdescribed hereinabove.

According to specific embodiments, the CXCR4 antagonistic peptide andthe immune-check point regulator of the invention can be administered toa subject in combination with other established or experimentaltherapeutic regimen to treat cancer including analgetics,chemotherapeutic agents, radiotherapeutic agents, hormonal therapy,immune modulators and other treatment regimens (e.g., surgery, celltransplantation e.g. hematopoietic stem cell transplantation) which arewell known in the art.

According to some embodiments of the invention the method furthercomprises administering a vaccine and optionally wherein the vaccine isan HPV vaccine.

According to a specific embodiment, the vaccine is a human papilomavirus (HPV, e.g., HPV 16 vaccine) typically targeting E6 and/or E7. Thevaccine may be a preventive vaccine or a therapeutic vaccine. Detailedexamples of HPV vaccines which can be used along with the presentteachings can be found in Lin et al. J Formos Med Assoc. 2010 Jan;109(1): 4-24; and Rice et al. Cancer Gene Therapy 22, 454-462.

According to a specific embodiment, the preventive vaccines utilize thecapsid proteins L1 and L2 as target antigens, inducing antibodies toneutralize and prevent entry of HPV into cells. Expression ofrecombinant Ll, the major component of the capsid, in various cell typesresults in spontaneous assembly of virus-like particles (VLPs), whichare immunologically and morphologically similar to HPV virions.

According to another specific embodiment, the vaccines is Gardasil™ orCervarix™ Gardasil is a quadrivalent vaccine containing recombinant L1VLPs for HPV genotypes 6, 11, 16 and 18 whereas the bivalent vaccineCervarix contains L1 VLPs for HPV-16 and 18.

According to another specific embodiment, the vaccine is a monovalentHPV-16 L1 vaccine with an aluminium hydroxyphosphate sulfate adjuvant.

Exemplary, non-limiting, therapeutic vaccines comprise HPV E6 and E7antigens. These represent ideal targets for therapeutic vaccines sincethese are constitutively expressed in HPV-infected cells and not healthycells. E6 and E7 are essential to the induction and maintenance ofcellular transformation, and thus are unlikely to be lost in an attemptto evade the immune system.

According to a specific embodiment, the therapeutic vaccines target E6and/or E7.

Therapeutic vaccines typically include:

Live vector vaccines—Vector-based vaccines can deliver the antigens E6and E7 to the dendritic cells (DCs), stimulating antigen expressionthrough MHC class I (to CD8+ cytotoxic T cells) and MHC class II (toCD4+ helper T cells). Viral vectors used adenovirus, adeno-associatedvirus, vaccinia virus and alphaviruses, such as the Venezuelan equineencephalitis (VEE) virus;

Peptide/protein-based vaccines—Administered peptides and proteinsderived from HPV antigens (e.g., E6 and/or E7) are taken up by DCs,processed and expressed via MHC II and/or Ito the appropriate CD4+/CD8+T cells;

Cell-based vaccines—dendritic cell-based or tumor cell based vaccines;and

Nucleic acid-based vaccines e.g., naked DNA based vaccines (e.g.,ZYC-101 and ZYC-101a), naked RNA replicon vaccines.

The CXCR4 antagonistic peptides and/or the immune-check point regulators(e.g. PD1 antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3antagonist, TIM-3 antagonist, KIR antagonist, IDO antagonist, OX40agonist, CD137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28agonist and ICOS agonist) described hereinabove can be administered tothe subject per se, or in a pharmaceutical composition where it is mixedwith suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the CXCR4 antagonisticpeptides and/or the immune-check point regulators accountable for thebiological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington' s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, intradermal, subcutaneous andintramedullary injections as well as intrathecal, directintraventricular, intracardiac, e.g., into the right or left ventricularcavity, into the common coronary artery, intravenous, intraperitoneal,intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

The CXCR4 antagonistic peptide of the invention, the immune-check pointregulator or the pharmaceutical composition comprising same can beadministered in the same route or in separate routes.

According to a specific embodiment, the CXCR4 antagonistic peptide ofthe invention or the pharmaceutical composition comprising same isadministered subcutaneously.

According to another specific embodiment, the CXCR4 antagonistic peptideof the invention or the pharmaceutical composition comprising same isadministered intravenously.

According to a specific embodiment, the immune-check point regulator orthe pharmaceutical composition comprising same is administeredintravenously.

According to a specific embodiment, the immune-check point regulator orthe pharmaceutical composition comprising same is administered via asubcutaneous route.

Pharmaceutical compositions of some embodiments of the invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodimentsof the invention thus may be formulated in conventional manner using oneor more physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to some embodiments of the invention are convenientlydelivered in the form of an aerosol spray presentation from apressurized pack or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes.

Aqueous injection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of theactive ingredients to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of some embodiments of the invention mayalso be formulated in rectal compositions such as suppositories orretention enemas, using, e.g., conventional suppository bases such ascocoa butter or other glycerides.

Alternative embodiments include depots providing sustained release orprolonged duration of activity of the active ingredient in the subject,as are well known in the art.

Pharmaceutical compositions suitable for use in context of someembodiments of the invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. More specifically, according to specific embodiments, atherapeutically effective amount means an amount of active ingredientseffective to prevent, alleviate or ameliorate symptoms of a disorder(e.g., cancer) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to providelevels of the active ingredient are sufficient to induce or suppress thebiological effect (minimal effective concentration, MEC). The MEC willvary for each preparation, but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Detection assays can beused to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

According to specific embodiments the CXCR4 antagonistic peptide of theinvention or the pharmaceutical composition comprising same isadministered in a dose ranging between 0.1 to10 mg/kg of body weight,between 0.1 to 2 mg/kg of body weight, between 0.1 to 1 mg/kg of bodyweight, between 0.3 to 10 mg/kg of body weight, between 0.3 to 2 mg/kgof body weight, between 0.3 to 1 mg/kg of body weight or between 0.3 to0.9 mg/kg of body weight.

According to a specific embodiment, the CXCR4 antagonistic peptide ofthe invention or the pharmaceutical composition comprising same isadministered in a dose ranging between 0.5-2.0 mg/kg.

According to specific embodiments, the immune-check point regulator orthe pharmaceutical composition comprising same is administered in a doseranging between 0.001 to 30 mg/kg body weight, between 0.001 to 20 mg/kgbody weight, between 0.001 to 10 mg/kg body weight, between 0.001 to 1mg/kg body weight, between 0.01 to 30 mg/kg body weight, between 0.01 to20 mg/kg body weight, between 0.01 to 10 mg/kg body weight, between 0.01to 1 mg/kg body weight, between 0.1 to 30 mg/kg body weight, between 0.1to 20 mg/kg body weight, between 0.1 to 10 mg/kg body weight, between0.1 to 1 mg/kg body weight, between 1 to about 30 mg/kg, between 1 toabout 20 mg/kg or between 1 to about 10 mg/kg.

The desired dose can be administered at one time or divided intosub-doses, e.g., 2-4 sub-doses and administered over a period of time,e.g., at appropriate intervals through the day or other appropriateschedule.

According to specific embodiments, the CXCR4 antagonistic peptide of theinvention, the immune-check point regulator or the pharmaceuticalcomposition comprising same is administered multiple times e.g. 2-10,over a period of time e.g. for several days to several weeks atappropriate intervals e.g. once a day, twice a week, once a week, onceevery two weeks, once a month, once every 3 to 6 months.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above.

According an aspect of the present invention there is provided anarticle of manufacture or a kit identified for use in treating cancer,comprising a packaging material packaging a peptide having an amino acidsequence as set forth in SEQ ID NO: 1 or an analog or derivative thereofand an immune-check point regulator (e.g. PD1 antagonist, PDL-1antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist, KIRantagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27 agonist,CD40 agonist, GITR agonist, CD28 agonist and ICOS agonist.

The peptide and the immune-check point regulator may be packaged in thesame container or in separate containers; each possibility represents aseparate embodiment of the present invention.

According to specific embodiments, the peptide and the immune-checkpoint regulator are in separate formulations.

According to other specific embodiments, the peptide and theimmune-check point regulator are in a co-formulation.

It is expected that during the life of a patent maturing from thisapplication many relevant immune-check point regulators will bedeveloped and the scope of the term “immune-check point regulator” isintended to include all such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, CT (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, CA (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Use of BL-8040 for Treating Cancer

BL-8040 is safe and well tolerated drug that was shown to induce rapidmobilization of hematopoietic stem/progenitor cells and mesenchymal stemcells as well as T cells, B cells, NK cells, NKT cells and ImDC to theperipheral blood. Therefore, BL-8040 can be used to induce themobilization and dissemination of immature DC, NK cells, B cells,monocytes/macrophages and T effector and memory cells into tumors.

Experimental Procedures

According to one protocol, BL-8040 is injected into a cancer patient ata dose of 0.5-2.0 mg/kg for 3-10 days and then 1-3 times a week incombination with immunomodulatory antibodies that antagonize with aninhibitory check point molecule such as anti-CTLA4 (such as produced byMedlmmne or BMS), anti-PD-1 (such as produced by BMY, AZY, Merck orCuretech), anti-PDL-1 (such as produced by Roche, Merck Serono, AZY orBMS), anti-LAG-3 (such as produced by BMS or Immutep), and anti-KIR(such as produced by BMY or Innate Pharma).

According to another protocol, BL-8040 is injected into a cancer patientat a dose of 0.5-2.0 mg/kg for 3-10 days and then 1-3 times a week incombination with a fusion protein targeting an inhibitory check pointmolecule such as PD1 (such as produced by AZY).

According to another protocol BL-8040 is injected into a cancer patientat a dose of 0.5-2.0 mg/kg for 3-10 days and then 1-3 times a week incombination with a small molecule that antagonizes with an inhibitorycheck point molecule such as IDO [such as NLG919 (produced by Roche),F001287 (produced by BMY), Indoximod (produced by NewLink Genetics),NLG-919 (produced by NewLink Genetics) and INCB-024360 (produced byIncyte)].

According to another protocol BL-8040 is injected into a cancer patientat a dose of 0.5-2.0 mg/kg for 3-10 days and then 1-3 times a week incombination with immunomodulatory antibodies that activates aco-stimulatory check point molecule such as CD40 (such as produced byPfizer or Seattle Genetics Inc.), 4-1BB (such as produced by BMY orMerck Serono), GITR (such as produced by Merck or GITR Inc.), OX40 (suchas produced by AZY or Roche), and CD27 (such as produced by Celldex).

According to another protocol, BL-8040 is injected into a cancer patientat a dose of 0.5-2.0 mg/kg for 3-10 days and then 1-3 times a week incombination with a fusion protein targeting a co-stimulatory check pointmolecule such as OX-40 (such as produced by AZY).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A method of treating a solid tumor in a humansubject in need thereof, the method comprising: (a) administering to thehuman subject a therapeutically effective amount of a peptide having anamino acid sequence as set forth in SEQ ID NO: 1; (b) administering tothe human subject a therapeutically effective amount of a PD1antagonist; and (c) administering to the human subject a therapeuticallyeffective amount of a chemotherapeutic agent, thereby treating the solidtumor in the human subject.
 2. The method of claim 1, wherein said solidtumor is pancreatic cancer.
 3. The method of claim 1, wherein said PD1antagonist is an anti-PD1 antibody.
 4. The method of claim 1, whereinsaid solid tumor is pancreatic cancer, wherein said PD1 antagonist is ananti-PD1 antibody and wherein said peptide is as set forth in SEQ ID NO:1.